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Ribosomal RNA Chrysopogon Aciculatus (Retz.) Trin
2557
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Ribosomal RNA Chrysopogon Aciculatus (Retz.) Trin
2557
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GENETIC COMPARISON OF RIBOSOMAL RNA OF Chrysopogon Aciculatus (Retz.) Trin
IN THAILAND
By
Miss Sumolthip Somboonchan
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree
Master of Science Program in Forensic Science
Graduate School, Silpakorn University
Academic Year 2014
Copyright of Graduate School, Silpakorn University
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“
Ribosomal RNA Chrysopogon Aciculatus (Retz.) Trin ”
……...........................................................
( . )
.......... .................... . ...........
.
....................................................
( . )
............/......................../..............
....................................................
( . )
............/......................../..............
....................................................
( . )
............/......................../..............
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53312333 :
: / Chrysopogon aciculatus (Retz.) Trin./
: Ribosomal RNA
Chrysopogon aciculatus (Retz.) Trin . : . .
. 94 .
Chrysopogon aciculatus (Retz.) Trin. 14
14
, , Chrysopogon aciculatus (Retz.) Trin.
(P<0.05)
E 8 ,
E 7 E 2
Chrysopogon aciculatus (Retz.) Trin.
........................................ 2557
........................................
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Chrysopogon aciculatus
Chrysopogon aciculatus
Chrysopogon aciculatus
Chrysopogon aciculatus
Chrysopogon aciculatus
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Ribosomal RNA Chrysopogon aciculatus (Retz.) Trin
.
.
.
,
,
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……………………………………………………………………………...
…………………………………………………………………………..
………………………………………………...……………………………
……………………………………………………..……………………………
………………………………………………..…………………………………….
1 …………………………………………………………..………………….……… 1
……….....………………….……….. 1
………………………………..…………………… 3
………………………..……………………………… 3
……………………...…………………………………... 4
……………………...……..………………… 4
………………………...………………………………... 5
……………………………………………………………… 5
…………………………………………………………... 6
2 ………………………………………………………… 7
………………………………………………... 7
………………………….………..……………………….. 11
(plant genome structure)………………………….. 13
…………… 18
………………….. 19
……………………………… 20
………………………………………………. 22
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(Polymerase chain reaction : PCR)……………... 26
…………………………………… 28
(contamination control) PCR…….…… 31
(DNA sequencing)………………….……. 32
dye terminator
Sequencing (Bigdye)…………………...………………………………….... 32
………….…………………………………………………………. 35
…………………………….…………………………….. 38
3 ……………………….………………………………………… 40
…………………………………………………….…………... 40
……………………………………………………….………... 40
…………………………………………………….…….. 42
………………………………………………….………… 42
4 ……………………………………………………..…………. 47
…………………………………………. 47
ribosomal RNA (rRNA) gene……………… 64
……………………………… 66
5 …………………………………………………... 74
……………………………………………………………… 74
………………………………………………………………….. 75
………………………………………………………………… 76
……………………………………………………………………………. 77
………………………………………………………………………………... 81
. …………………………………….………. 82
. ……………………………………... 84
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. primer………………… 87
. ……………………………………………………… 91
……………………………………………………………………………... 94
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2.1 …………….. 3 .
………………………………………………………………………….. 0 4. ………...……………………………………. 48 4. ..………………….... 55 4.3 rRNA gene
4 …………….…………………………………………………….. 7
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2.1 , -
……………………………………...…………………………. 7 . …………………………………………………… 8 .3 ……………….. 0 .4 (DNA)………………………………… .5 …………...……………………………………………. 6 .6 Trichoplax adhaerens………………………………. 7 .7 green alga Nephroselmis…………………………….. 7 .8 ………………………….. 8 .9 …………...………………………………..
2.1 semiconservative……………...……………….. 4 . DNA polymerase…………………………………..…………….. 5 . DNA polymerase Okazaki fragment…..………. 6 . 3 PCR..………... 30 . 4 PCR……………………..…………………………………….. 30 . 5 dideoxynucleoside triphosphate…………..………………………... 33 . 6 dye terminator sequencing…………..……………………………………... 34 . 7 …………………………………………………………………….... 36
2.1 …………………………………………………………...... 37 4. ……………………………………………... 5 4. ……………………... 59
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4.3 DNA Agarose gel……………………………………………………………… 65 4.4 rRNA gene ……………….. 7 4.5 Phylogenetic tree 4 …. 73
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1
1
1.
10 . . 2545 – 2554
45,402 . . 2545 30,130 . . 2554 5
, , ,
( , 2555)
, , ,
(crown)
( , 2535)
, ,
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2
10,000 650
Chrysopogon aciculatus (Retz.) Trin.
,
, bard
DNA DNA
DNA chloroplast
DNA DNA 120-
220 chloroplast DNA 40 chloroplast DNA
Poaceae haploid
DNA diploid DNA
DNA
287,655 258,650
(lnternational Union for Conservation of Nature and Natural Resources, 2006)
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3
DNA DNA primer
Chrysopogon aciculatus (Retz.)
Trin.
2.
2.1 Chrysopogon aciculatus (Retz.)
Trin. , ,
2.2
Chrysopogon aciculatus (Retz.) Trin.
2.3 primer DNA
PCR
3.
3.1 Chrysopogon aciculatus (Retz.) Trin.
3.2 Chrysopogon aciculatus (Retz.) Trin.
chloroplast DNA
3.3 primer PCR DNA
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4.
4.
Chrysopogon aciculatus (Retz.) Trin.
, , 14
3
4.2 1 . . 2555 1
. . 2556
4.3 1
4.4 DNA plant
DNA extraction DNA polymerase chain reaction (PCR)
dye terminator sequencing
4.5
Alignment Phylogenetic tree
SPSS (One-way Anova)
5.
chloroplast DNA DNA
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5
6.
6.1
. . 2555 . . 2556
silica gel
6.2
7.
Gene
chloroplast DNA
Polymerase Chain Reaction (PCR) DNA
(cloning)
DNA sequencing
Transition
Transversion
Poaceae (Gramineae) Chrysopogon
aciculatus (Retz.) Trin. , , ( ),
, ( ) ( ) Love Grass, Golden
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beardgrass, Grass seed, Mackie’s pest, Needle grass, Pilipiliula
8.
8.1
8.2 chloroplast DNA
DNA sequencing
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2
(DNA)
(deoxyribonucleic acid)
(phosphate group) 5 (5-carbon sugar)
(nitrogen base)
2.1 ,
: Thaibiotech.info, (Chemical Composition of DNA),
Accessed May 23, 2013. Available from http://www.thaibiotech.info/chemical-composition-of-
dna.php
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(monophosphate)
(diphosphate) (triphosphate)
(deoxyribose)
(ribose)
2
(purine) (adenine, A) (guanine,G)
2
(pyrimidine) (thymine,T)
(cytosine,C)
(uracil,U) ( , 2553)
2.2
: National Human Genome Research Institute (NHGRI) Glossary of Genetic Terms,
Nucleotide, Accessed April 17, 2013. Available from
http://www.ncbi.nlm.nih.gov/Class/MLACourse/Original8Hour/Genetics/nucleotide.html
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(X-ray diffraction)
5´-phosphate 3´-OH (double helix)
2 (antiparallel)
5´-phosphate 3´-OH 3´-OH 5´-phosphate
A
T 2 G C 3
A T
major groove minor groove
3.4 (A0 : Angstrom
0.34 x 109 ) Watson Crick . .
1953 ( , 2552)
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2.3
: National Human Genome Research Institute (NHGRI) Glossary of Genetic Terms,
Nucleotide, Accessed April 17, 2013. Available from
http://www.ncbi.nlm.nih.gov/Class/MLACourse/Original8Hour/Genetics/nucleotide.html
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2.4 (DNA)
: Peter Koeppel, Nucleotides – building blocks of life, Accessed April , 2013. Available
from http://www.nucleotidenutrition.com/nucell-im-featured-in-the-article-nucleotides-the-
building-blocks-of-life/
1
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(nucleoprotein) 2
1. (histone protein)
2. (nonhistone protein)
transcription factors
(gene) 3%
30,000-50,000
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2.1
(Mb)
Hepatitis B virus
HIV-1
Bacteriophage I
Escherichia coli S. cerevisiae (yeast)
C. elegans (nematode)
D. melanogaster (fruit fly)
Mus musculus (mouse)
0.0032
0.0092
0.0485
4.6392
12.155
97
137
3,000
4
9
80
4,400
6,300
19,000
13,600
20,000-30,000
: , ( :
, 2552), 27.
97% (junk DNA)
noncoding DNA noncoding gene
intron, promoters, leader/trailers pseudogenes (
) gene fragments
extragenic DNA
(single copy or very low copy number)
(repetitive sequence) ( , 2552)
(plant genome structure)
(nuclear genome) (organelle genome)
2 (chloroplast genome)
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(chloroplast DNA cpDNA) (mitochondria genome)
(mitochondria DNA mtDNA)
(genic DNA)
(nongenic DNA)
95%
(repetitive DNA)
2
1. (interspersed repeats) (repeat unit)
2
SINES (short interspersed elements) 100-
150
LINES (long interspersed elements) 500
2. (tandemly repeats)
(length)
(replication copy)
(sattellite) 2
100-300 1,000-100,000
(centromere)
(minisattellite) 10-60
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(microsatellite) 1-6
100 simple sequence
repeats (SSR), short tandem repeats (STR) simple repetition
sequences (SRS)
(linear)
10 125,000
(ploidy)
(double stranded circular DNA)
180-2500
16
(respiration) (oxidation phosphorylation)
ATP
120-220
(photosynthesis)
(inverted repeat) 2
( , 2553)
(gymnosperm)
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Pinaceae Taxodiaceae
2.5
: NUCLEIC ACIDS A: THE STRUCTURE OF DNA, Accessed April 17, 2013.
Available http://employees.csbsju.edu/hjakubowski/classes/ch331/dna/oldnastructure.html
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2.6 Trichoplax adhaerens
: Stephen L. Dellaporta, Mitochondrial genome of Trichoplax adhaerens supports Placozoa
as the basal lower metazoan phylum, Accessed April 17, 2013. Available
http://www.pnas.org/content/103/23/8751/F1.expansion.html
2.7 green alga Nephroselmis
: Martin F. Wojciechowski, Structure of plant chloroplast genome, Accessed April 17,
2013. Available http://tolweb.org/notes/?note_id=4148
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2
(endosymbiont hypothesis)
-purple bacteria
1,200
ATP
cyanobacteria
2.8
: Kenneth Todar, Todar's Online Textbook of Bacteriology, Accessed April 17, 2013.
Available http://textbookofbacteriology.net/themicrobialworld/origins.html
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(uniparental inheritance)
(haploid)
(haplotype)
(angiosperm)
75% 25%
(gymnosperm) Pinaceae Taxodiaceae
3 4-5
Pinaceae
( , 2552)
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2.2
: , : ( :
, 2552), 13
1. (ribosomal DNA)
(coding region)
(repeat unit)
10%
intergenic spacer (IGS)
(transcribed region) external transcribed spacer (ETS) small rDNA
18S, 1(internal transcribed spacer ITS1), 5.8S, 2
(ITS2), 26S large rDNA large rDNA 25S 28S
S (Svedberg unit)
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2.9
: , ( :
, 2553), 19.
18S, 5.8S 26S (ribosomal rRNA gene)
(mRNA) (tRNA)
18S, 5.8S 26S 18S
1,800 5.8S 160 26S 3,300
IGS 1-8
(conserved region)
18S, 5.8S 26S ITS IGS
2. trnT, trnL trnF
trnT, trnL trnF coding region
tRNA (threonine: T), (leucine: L)
(phenylalanine: F)
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(secondary structure) (anticodon)
trnT, trnL trnF trnT, intergenic
spacer trnT trnL 5´ exon, trnL intron, intergenic spacer
trnL 3´ exon trnF
intergenic spacer
( , 2553)
2
(semiconservative)
1 1
5´ 3´
(template) 3´
5´ 5´ 3´ (leading strand)
5´ 3´
Okazaki fragment
(lagging strand)
1. helix-destabilizing protein helicase
ATP
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2. single strand DNA binding protein (SSB DBP)
nuclease
3. DNA gyrase topisomerase (replication fork)
4. primase (primer)
(DNA
polymerase) primase
E.coli 1 60,000 50-100
1 primosome
5. DNA polymerase
3´(-OH) 5´ 3´
E.coli DNA polymerase 3 DNA polymerase I, II III (pol I,
pol II, pol III) DNA polymerase I II 1
DNA polymerase III 3
4 dTTP dATP dGTP dCTP
pol I
(polymerization) 2
5´ 3´ exonuclease 3´ 5´ exonuclease
pol I
(proofreading)
3´ 5´ exonuclease
pol I (RNA
primer) 5´ 3´ exonuclease
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pol I 2 -
polymerization 3´ 5´ exonuclease Klenow fragment
5´ 3´ exonuclease
pol II 5´ 3´ exonuclease
3´ polymerase
polymerase III pol III
5´ 3´ exonuclease
pol I pol III
5´ Okazaki
6. DNA ligase
ligase
5´-phosphate 3´-hydroxyl
(nick)
2.10 semiconservative
: Encyclopædia Britannica, Inc., double helix: DNA structure, Accessed April 17, 2013.
Available http://kids.britannica.com/elementary/art-107613/The-structure-of-DNA-is-a-double-
helix
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2.11 DNA polymerase
: Bradoo, Sapna D., and Anja-Katrin Bielinsky, DNA Replication and Checkpoint Control
in S Phase. Accessed April 17, 2013. Available http://www.nature.com/scitable/topicpage/dna-
replication-and-checkpoint-control-in-s-14202419
helicase
single strand DNA binding protein
DNA gyrase
primase DNA polymerase III
3´
DNA polymerase III
Okazaki fragments 1,000-
2,000 Okazaki DNA polymerase I
5´ 3´ exonuclease
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3´ DNA polymerase III
3´
(nick translation) DNA
ligase ( , 2545)
2.12 DNA polymerase Okazaki fragment
: DNA as the Genetic Material, Accessed April 17, 2013. Available
http://home.earthlink.net/~dayvdanls/dna1.htm
(Polymerase chain reaction : PCR)
Kary Mullis . . 1986
DNA polymerase
(DNA
primer) 90 C
- -
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(1-10 )
(DNA template)
(DNA primer) 5´
3´
dATP, dTTP, dGTP dCTP
(DNA polymerase)
Mg2+ Mn2+ (
, 2552)
20-100 0.2 (thin
wall)
DNA thermal cycler
(PCR product)
( , 2553)
3
1. (DNA denaturation)
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2
90oC
(% GC content)
Melting temperature (Tm)
14
Tm = 4oC x ( G C ) + 2oC x (
A T )
14
Tm = 64.9oC + 41oC x ( G C – 16.4) / N
N =
2. (Annealing)
2
5´ 3´
5´ 3´ sense-primer forward primer
3´ ( 5´
3´ ) antisense primer reverse primer
(annealing) annealing
temperature 52-58oC melting temperature (Tm)
108
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Annealing temperature (Ta)
Ta = 0.3 x Tm(primer) + 0.7 Tm(product) – 14.9
Tm(primer) = melting temperature
Tm(product) = melting temperature
Tm primer 5oC
3. (Extension)
3´
(dATP, dTTP, dGTP dCTP)
3´
Taq DNA polymerase Thermus aquaticus
yellow stone
60oC 90oC
denaturation-annealing-extension
30 230 109
20-35 PCR
Taq DNA polymerase
proofreading (3´ 5´ -exonuclease activity ) PCR
30
1 mismatch 2,000-3,000 ( , 2552)
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2.13 1 PCR
: Hernandez, Jennifer., Profile of hot start PCR reaction, Accessed April 17, 2013.
Available http://jenniferhernandez.wordpress.com/
2.14 PCR
: Department of Chemistry, University of Warwick, PCR , Accessed April 17, 2013.
Available http://www2.warwick.ac.uk/fac/sci/chemistry/research/arodger/arodgergroup/people/
msrhab/ research/pcr/
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(contamination control) PCR
PCR
PCR (negative control)
(template-negative)
PCR (positive control)
PCR
PCR
(short spin) 5-10
distilled/deionized
master mixed
(Ziploc)
(disposable) PCR (tip)
(powder free gloves)
( , 2553)
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(DNA sequencing)
Sanger
(poly-acrylamide gel electrophoresis)
dye terminator sequencing (Bigdye)
3´-
OH
3´,5´- 3´-OH
3´,5´-
3´-OH
(dideoxynucleoside triphosphate; ddNTP) ddATP, ddGTP, ddCTP, ddTTP
Klenow fragment Bacteriophage T7 (T7 DNA
polymerase) Tag DNA polymerase Klenow fragment C-
terminal DNA polymerase 5´ 3´ exonuclease activity
( N-terminal
5´ 3´ exonuclease activity )
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2.15 dideoxynucleoside triphosphate
: Pharmacia Biotech Inc., DNA Sequencing, Accessed April 17, 2013. Available
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html
ddNTP
ddNTP fluorescence
base
polyacrylamide gel electrophoresis ( , 2552) sequence
DNA sequencing ( , 2544)
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2.16 dye terminator sequencing
: Mammalian Genetics University of Florida, DNA Sequencing Facilities Center for
Mammalian Genetics University of Florida, Accessed April 19, 2015. Available
http://www.dna-sequencing-service.com/dna-sequencing/dna-sequencing/
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: Poaceae (Gramineae)
: Chrysopogon aciculatus (Retz.) Trin.
: , , ( ), ,
( ) ( ) ( , 2553)
: Love Grass, Golden beardgrass, Grass seed, Mackie’s pest, Needle
grass, Pilipiliula
20-60 .
2-9.5
. 7-12 .
0.1-0.2 .
(panicle) 16-30 .
4 .
3 3.5-4 .
2.5-3.5 . 2
2.5-3 .
2.5-3 . 5 . 1 1.5 .
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2 3
1.3 . 1.1 . 2
5.7
. 2-2.5 . 5 .
3 4.5 . 1 2
3 .
3 .
1.8 . 2 3
0.5 . 2.2 .
(caryopsis) 2.3 .
(Ianceolate)
0.3-0.5 . 1.8-2.0 . ( , 2544)
2.17
: Warm Season Turfgrasses, Accessed April 17, 2013. Available
http://www.toyogreen.com/landscape/Turfing.html
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2.18
: Julia Scher, Forestry Images, Accessed April 17, 2013. Available http://www.forestryimages.org/browse/detail.cfm?imgnum=5376365
26 9 Chrysopogon
aciculatus (Retz.) Trin, Chrysopogon festicoides (Presl) Veldk., Chrysopogon fulvus (Spreng.)
Choiv., Chrysopogon gryllus (L.) Trin., Chrysopogon lawsonii (Hook. f. ) Veldk.,
Chrysopogon orientalis (Desv.) A. Camus, Chrysopogon perlaxus Bor, Chrysopogon serrulatus
Chrysopogon zizanioides (L.) Roberty (S-Si)
(
, 2553)
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( ,
2548)
bard
( , 2544)
( ,
2535) ( , 2548)
Lian Lay Hoong Koh Chong Lek, 2005 DNA
hypervariable (HV) 3 HV1, HV2 HV3
noncoding control region DNA (mtDNA) DNA
195 ( 13 15 )
HV1, HV2 HV3 noncoding control region
Li-Chin Tsai , 2006 DNA trnL intron
trnL-trnF IGS 373 80
, 206 269 trnL intron
trnL-trnF IGS
20 Blind testing 15 9
99.4% 6 GenBank EMBL
Kathleen et al, 2007 sand live oak (Quercus geminate)
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sand live oak DNA
microsatellites
4 2.06 x 10 -6 DNA
PCR DNA
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3
1.
14
1 , , , , , , ,
, , , , , 3
27 . . 2555 7 . .2556
2.
2.1
2.1.1 Microcentrifuge tube, 1.5 ml.
2.1.2 PCR tube, 0.5 ml.
2.1.3 Beaker, 50 ml.
2.1.4 Flasks, 250 ml.
2.1.5 Volumetric flasks, 100 ml.
2.1.6 Cylinder, 100 ml., 1,000 ml.
2.1.7 Microcentrifuge tube racks
2.1.8 Parafilm
2.1.9 Autopipettes
2.1.10 Water bath
2.1.11 Microcentrifuge
2.1.12 Autoclave
2.1.13 Hot air oven
2.1.14 Freezer -20°C
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2.1.15 Microwave
2.1.16 Vortex
2.1.17 Incubator
2.1.18 Agarose chamber
2.1.19 Electrophoresis chamber
2.1.20 Power supply
2.1.21 UV transluminator
2.1.22 Thermal cycler
2.1.23 Light microscope
2.1.24
2.1.25
2.1.26
2.1.27
2.1.28
2.2
2.2.1 Ethanol 70%
2.2.2 Absolute ethanol
2.2.3 Proteinase K
2.2.4 R Nase A
2.2.5 1X TAE buffer
2.2.6 Agarose
2.2.7 6X loading dye
2.2.8 100 bp DNA Ladder (DNA Marker)
2.2.9 10X PCR buffer
2.2.10 50 mM MgCl2
2.2.11 dNTP mixed (dATP, dCTP, dTTP, dGTP)
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2.2.12 Taq DNA polymerase 5 unit/ l.
2.2.13 ddH2O
2.2.14 DNA Primer
2.2.15 Sterile water
2.2.16 Ethidium bromide
2.2.17 Liquid nitrogen
2.2.18 Silica gel
2.2.19 GF-1 plant DNA extraction Kit
2.2.19.1 Buffer PL
2.2.19.2 Buffer PB
2.2.19.3 Wash Buffer
2.2.19.4 Elution Buffer
2.2.20 Gel/PCR DNA Fragments Extraction Kit
2.2.20.1 DF Buffer
2.2.20.2 Wash Buffer
2.2.20.3 Elution Buffer
3.
4.
4.1
(Convenience Accidental Sampling)
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4.2 ,
,
,
( , 2551)
Olympus SZ51
4.3 primer DNA PCR
primer3 Genbank GQ . GI:
ribosomal RNA (rRNA) gene chloroplast DNA
4.4 GF-1 plant DNA extraction
Kit
4.4.1 10-30
microcentrifuge
4.4.2 Buffer PL 280 l. vortex 30
4.4.3 Proteinase K 20 l. 65°C 1-2
4.4.4 centrifuge 14,000-16,000 g 5
microcentrifuge RNase A 5 l. 37°C 5
4.4.5 Buffer PB 640 l. -
65°C 10
4.4.6 Absolute ethanol 200 l.
4.4.7 column centrifuge 10,000 g 1
4.4.8 column wash Buffer 750 l. centrifuge
10,000 g 1
4.4.9 column microcentrifuge Elution Buffer
50 l. column membrane 2
สำนกัหอ
สมุดกลาง
44
4.4.10 centrifuge 10,000 g 1
-20°C
4.5 PCR
4.5.1 PCR tube 10X PCR buffer, 50 mM MgCl2 , 10 mM
dNTP, 25 pmole/ l. forward primer, 25 pmole/ l. reward primer, 0.5U Taq DNA polymerase,
ddH2O template DNA 50 l.
4.5.2 PCR tube Thermal cycler
PCR cycle
1st denaturation 94°C 5
Denaturation 94°C 1
Annealing 53°C 1
Extension 72°C 30
Final extension 72°C 7
Final step 4°C
35
4.6 (Gel Electrophoresis)
4.6.1 1% agarose agarose 0.4 1X TAE
40 ml. agarose agarose chamber
4.6.2 agarose electrophoresis chamber 1X TAE
agarose
สำนกัหอ
สมุดกลาง
45
4.6.3 6X loading dye 2 l. parafilm product PCR
4 l. 6X loading dye parafilm
agarose
4.6.4 100 bp DNA Ladder DNA marker 1 l.
agarose
4.6.5 100 V 75 mA 40
4.6.6 agarose 1% ethydium bromide
15
4.6.7 UV transluminator
4.7 agarose UV
4.8 agarose Gel/PCR DNA Fragments Extraction
Kit
4.8.1 agarose microcentrifuge DF buffer
500 l. vortex
4.8.2 microcentrifuge 60°C 15
agarose 2-3
4.8.3
4.8.4 DF column 2 ml. Collection Tube DF
column centrifuge 14,000 – 16,000 g 30 column
DF column 2 ml. Collection Tube
4.8.5 wash buffer 600 l. DF column 1
สำนกัหอ
สมุดกลาง
46
4.8.6 centrifuge 14,000 – 16,000 g 30
4.8.7 wash buffer 4.8.5 4.8.6
4.8.8 centrifuge 14,000 – 16,000 g 3 column
4.8.9 DF column microcentrifuge Elution buffer
DF column 2 Elution buffer
4.8.10 centrifuge 14,000 – 16,000 g 2
4.9 DNA sequencing dye terminator sequencing
4.10 Phylogenetic tree Alignment
MEGA 5.1
สำนกัหอ
สมุดกลาง
47
4
1.
14
14
E01 0.4 E06
1.2
(p<0.05)
S01 , S02 , E01
, E02 E03
0.05 E04 0.2
(p<0.05)
E02 2.65 E05
20.4
(p<0.05)
S01 E03
0.3 N01 0.9
(p<0.05)
S02 3.2
สำนกัหอ
สมุดกลาง
48
C01 11.3
(p<0.05)
S01
17.2 N01 81.50
(p<0.05)
4.1 4.1
4.1
( . .)
( . .)
( . .) ( . .)
( . .) ( . .)
S01
sum 1.6 0.15 11.6 1.25 17.1 64.8
Average 0.533333 0.05 3.866667 0.416667 5.7 21.6
count No. 3 3 3 3 3 3
Max 0.55 0.05 4 0.5 6.7 24.5
Min 0.5 0.05 3.6 0.3 4.7 17.2
SD 0.02357 6.93889E-18 0.188562 0.084984 0.816497 3.163332
S02
sum 1.7 0.15 11.4 1.4 12.6 73.5
Average 0.566667 0.05 3.8 0.466667 4.2 24.5
count No. 3 3 3 3 3 3
Max 0.6 0.05 4 0.6 4.8 29
Min 0.5 0.05 3.6 0.4 3.2 18.2
SD 0.04714 6.93889E-18 0.163299 0.094281 0.711805 4.589118
S03
sum 2.85 0.3 18.6 1.7 24.7 147.5
Average 0.95 0.1 6.2 0.566667 8.233333 49.16667
count No. 3 3 3 3 3 3
Max 1 0.1 7.55 0.6 9 50
Min 0.85 0.1 4.1 0.5 7.6 48.6
SD 0.070711 1.38778E-17 1.504992 0.04714 0.579272 0.601849
สำนกัหอ
สมุดกลาง
49
4.1 ( )
( . .)
( . .)
( . .) ( . .)
( . .) ( . .)
E01
sum 1.4 0.15 14.6 1.55 12.2 69.5
Average 0.466667 0.05 4.866667 0.516667 4.066667 23.16667
count No. 3 3 3 3 3 3
Max 0.5 0.05 5.4 0.6 4.3 29
Min 0.4 0.05 4.3 0.45 3.9 19.9
SD 0.04714 6.93889E-18 0.449691 0.062361 0.169967 4.134677
E02
sum 2 0.15 8.4 1.55 19.7 146.2
Average 0.666667 0.05 2.8 0.516667 6.566667 48.73333
count No. 3 3 3 3 3 3
Max 0.7 0.05 3.05 0.6 7.2 59.5
Min 0.6 0.05 2.65 0.4 5.9 40
SD 0.04714 6.93889E-18 0.177951 0.084984 0.531246 8.089637
E
sum 2.15 0.15 13.2 1.25 15.5 88.4
Average 0.716667 0.05 4.4 0.416667 5.166667 29.46667
count No. 3 3 3 3 3 3
Max 0.8 0.05 5.2 0.5 6.1 35
Min 0.6 0.05 3.4 0.3 4.2 25.2
SD 0.084984 6.93889E-18 0.748331 0.084984 0.77603 4.099864
E04
sum 2.7 0.5 15.7 1.5 17.4 96
Average 0.9 0.166666667 5.233333 0.5 5.8 32
count No. 3 3 3 3 3 3
Max 0.95 0.2 6.1 0.6 7.2 40.4
Min 0.85 0.15 4.5 0.45 4.9 25.2
SD 0.040825 0.023570226 0.659966 0.070711 1.003328 6.307667
สำนกัหอ
สมุดกลาง
50
. ( )
( . .)
( . .)
( . .) ( . .)
( . .) ( . .)
E05
sum 2.85 0.4 33.75 1.6 24.15 182.3
Average 0.95 0.133333333 11.25 0.533333 8.05 60.76667
count No. 3 3 3 3 3 3
Max 1 0.15 20.4 0.6 8.85 73
Min 0.9 0.1 4.85 0.45 7.3 54.2
SD 0.040825 0.023570226 6.639403 0.062361 0.633772 8.658073
E06
sum 3.3 0.3 20.7 2.05 24.2 161.1
Average 1.1 0.1 6.9 0.683333 8.066667 53.7
count No. 3 3 3 3 3 3
Max 1.2 0.1 7.65 0.7 9.8 63.7
Min 1 0.1 6.4 0.65 6.9 45.4
SD 0.08165 1.38778E-17 0.540062 0.02357 1.249889 7.567034
E07
sum 2.5 0.3 12.1 1.5 22 125.8
Average 0.833333 0.1 4.033333 0.5 7.333333 41.93333
count No. 3 3 3 3 3 3
Max 0.9 0.1 4.4 0.6 7.6 43.6
Min 0.8 0.1 3.7 0.4 7 39.4
SD 0.04714 1.38778E-17 0.286744 0.08165 0.249444 1.820867
E08
sum 2.55 0.3 12.95 1.4 21.25 143.3
Average 0.85 0.1 4.316667 0.466667 7.083333 47.76667
count No. 3 3 3 3 3 3
Max 0.9 0.1 5.2 0.5 8.9 67.1
Min 0.8 0.1 3.15 0.45 6.05 37.7
SD 0.040825 1.38778E-17 0.860555 0.02357 1.288625 13.67463
สำนกัหอ
สมุดกลาง
51
. ( )
( . .)
( . .)
( . .) ( . .)
( . .) ( . .)
C
sum 2.7 0.3 43.2 1.2 29.5 143.8
Average 0.9 0.1 14.4 0.4 9.833333 47.93333
count No. 3 3 3 3 3 3
Max 1 0.1 15.5 0.45 11.3 59.7
Min 0.8 0.1 13.5 0.35 9 40.6
SD 0.08165 1.38778E-17 0.828654 0.040825 1.040299 8.4041
N01
sum 2.55 0.35 41.3 2.2 27.3 206.6
Average 0.85 0.116666667 13.76667 0.733333 9.1 68.86667
count No. 3 3 3 3 3 3
Max 1 0.15 20 0.9 9.8 81.5
Min 0.7 0.1 8.4 0.6 8.1 55.3
SD 0.122474 0.023570226 4.775167 0.124722 0.725718 10.71645
N02
sum 2.1 0.3 9.8 1.5 20.6 101.6
Average 0.7 0.1 3.266667 0.5 6.866667 33.86667
count No. 3 3 3 3 3 3
Max 0.8 0.1 3.6 0.5 7.1 38.1
Min 0.6 0.1 2.7 0.5 6.7 25.6
SD 0.08165 1.38778E-17 0.402768 0 0.169967 5.845986
สำนกัหอ
สมุดกลาง
52
( ) ( ) ( )
( ) ( ) ( )
4.1
( ) S01 ( ) S02
( ) S03 ( ) E01
( ) E02 ( ) E03
สำนกัหอ
สมุดกลาง
53
( ) ( ) ( )
( ) ( ) ( )
.
( ) E04 ( ) E05
( ) E06 ( ) E07
( ) E08 ( ) C01
สำนกัหอ
สมุดกลาง
54
( ) ( )
.
( ) N01 ( ) N02
Olympus SZ51
E03, E04, E05 N01
S01, S02 N02 S01 S02
S03, E05, E06 E08
Olympus SZ51
N01 N02
E08
E07
E02 4.2 4.2
สำนกัหอ
สมุดกลาง
55
4.2
S01 -
-
-
-
-
-
-
-
S -
-
-
-
-
-
-
-
S -
-
-
-
-
-
-
-
สำนกัหอ
สมุดกลาง
56
4.2 ( )
E01 -
-
-
-
-
-
-
-
E02 -
-
-
-
-
-
-
-
E -
-
-
-
-
-
-
-
สำนกัหอ
สมุดกลาง
57
4.2 ( )
E04 -
-
-
-
-
-
-
-
E05 -
-
-
-
-
-
-
-
E -
-
-
-
-
-
-
-
-
สำนกัหอ
สมุดกลาง
58
4.2 ( )
E07 -
-
-
-
-
-
-
-
E08 -
-
-
-
-
-
-
-
C01 -
-
-
-
-
-
-
-
สำนกัหอ
สมุดกลาง
59
4.2 ( )
N01 -
-
-
-
-
-
-
-
N02 -
-
-
-
-
-
-
-
( )
4.2
( ) S01
สำนกัหอ
สมุดกลาง
60
( )
( )
( )
4.2
( ) S02 ( ) S03
( ) E01
สำนกัหอ
สมุดกลาง
61
( )
( )
( )
4.2
( ) E02 ( ) E03
( ) E04
สำนกัหอ
สมุดกลาง
62
( )
( )
( )
4.2
( ) E05 ( ) E06
( ) E07
สำนกัหอ
สมุดกลาง
63
( )
( )
( )
4.2
( ) E08 ( ) C01
( ) N01
สำนกัหอ
สมุดกลาง
64
( )
4.2
( ) N02
2. ribosomal RNA (rRNA) gene
primer Chrysopogon aciculatus (Retz.) Trin.
primer3 ribosomal RNA (rRNA) gene
Genbank GQ856352.1 GI: 299831199 693
bp (Neamsuvan,O., Veldkamp,J.F., and Seelanan,T, 2009 ) primer
rRNA gene Forward sequence CCTTAGGCGTCAAGGAACAC
rRNA gene Reverse sequence TGCTTAAACTCAGCGGGTAG
primer DNA 512 bp
primer 2 C01 E03
DNA GF-1 Plant DNA Extraction Kit DNA
PCR primer primer3
สำนกัหอ
สมุดกลาง
65
PCR 1 cycle st denaturation 94°C
Denaturation 94°C
Annealing 53°C
Extension 72°C
Final extension 72°C
Final step 4°C
DNA
agarose gel DNA bp DNA Ladder DNA marker
primer ribosomal RNA gene DNA
2 DNA
primer ribosomal RNA gene PCR
4.3 DNA Agarose gel 1 DNA marker 2 C01
primer rRNA gene 3 E03 primer rRNA
gene
สำนกัหอ
สมุดกลาง
66
3.
14 DNA GF- Plant DNA
Extraction Kit DNA primer ribosomal RNA gene
512 bp
14 192 – 657 bp
465 bp
CLUSTAL O(1.2.1) multiple sequence alignment
EO7 AACGTGCT-GACGTA-CGCTCTATCTGAATAGGG-ACACTCC-GTAGCGATGACATCTTA 56
EO1 GGTCGTCTCGAGGTGACGATCGG-CTGC-TTCGGCTCCCACAGGCAGGGATGACATCTTA 58
EO8 CTCGTGCTCGACGGACGGTCGGCCTGCCTAGCCGCTCATCGCCGTAGCGATGACATCTTA 60
EO2 GGGGGTGCTCGACGGACGGTCGGCCTGCCTTCGGCT-CCCGCAGCAGCGATGACATCTTA 59
E03 TGCGAGCTCGGCGGAGCGGTCGGCCTGCCTTCGGCTCCCCGCAGCAGCGATGACATCTTA 60
E06 AGCGTGCT-GGCGGA-CGGTCGGCCTGCCTTCGGCTCCCCGCAGCAGCGATGACATCTTA 58
S02 ATC-TGCT-GGCGGA-CGGTCGGCCTGCCTTCGGCTCCCCGCAGCAGCGATGACATCTTA 57
S01 TGGATTCT-GGCGGA-CGGTCGGCCTGCCTTCGGCTCCCCGCAGCAGCGATGACATCTTA 58
E05 GAGGTGCTCGGCGGA-CGGTCGGCCTGCCTTCGGCTCCCCGCAGCAGCGATGACATCTTA 59
N02 GAGCTGCTCGGCGGA-CGGTCGGCCTGCCTTCGGCTCCCCGCAGCAGCGATGACATCTTA 59
C01 GTTCTGCTCGGCGGA-CGGTCGGCCTGCCTTCGGCTCCCCGCAGCAGCGATGACATCTTA 59
N01 AGCGTGCTCGGCGGA-CGGTCGGCCTGCCTTCGGCTCCCCGCAGCAGCGATGACATCTTA 59
E04 TTCGTGCTCGGCGGA-CGGTCGGCCTGCCTTCGGCTCCCCGCAGCAGCGATGACATCTTA 59
SO3 GGTCGTGCTCGGCGGACGGTCGGCCTGCCTTCGGCTCCCCGCAGCAGCGATGACATCTTA 60
* * * ** ************
EO7 ATCATCACGACTCTCGCCAACGGATATCTCGGCTCACGCATCGATGAAGAACGTAGCAAA 116
EO1 ATCCACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 118
EO8 TTCAACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 120
EO2 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 119
E03 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 120
สำนกัหอ
สมุดกลาง
67
E06 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 118
S02 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 117
S01 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 118
E05 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 119
N02 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 119
C01 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 119
N01 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 119
E04 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 119
SO3 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 120
** *********** ****************** ************************
EO7 ATGCGATACCTGGTGTTCCTTGCCGACTACCGCGAACCATCGAGTTTTTGAACGCAAGTT 176
EO1 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 178
EO8 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 180
EO2 ATGCGATACCTGGTGTGAATTGCCGACTCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 179
E03 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 180
E06 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 178
S02 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 177
S01 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 178
E05 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 179
N02 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 179
C01 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 179
N01 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 179
E04 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 179
SO3 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 180
**************** **** ** * *******************************
EO7 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 236
EO1 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTAGGCGTCACGCCAAAAGACACTCC 238
EO8 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 240
EO2 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 239
สำนกัหอ
สมุดกลาง
68
E03 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 240
E06 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 238
S02 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 237
S01 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 238
E05 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 239
N02 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 239
C01 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 239
N01 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 239
E04 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 239
SO3 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 240
************************************ ***********************
EO7 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 296
EO1 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGTAGGGCGCGGTGGG 298
EO8 CAACCCACCCACGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 300
EO2 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 299
E03 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 300
E06 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 298
S02 CAACCCACCCACGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 297
S01 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 298
E05 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 299
N02 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 299
C01 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 299
N01 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 299
E04 CAACCCACCCACGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 299
SO3 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 300
*********** ********************************** *************
EO7 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 356
EO1 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGTACGTGGTGGGCGACACCTAGT 358
EO8 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 360
สำนกัหอ
สมุดกลาง
69
EO2 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 359
E03 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 360
E06 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 358
S02 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 357
S01 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 358
E05 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 359
N02 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 359
C01 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 359
N01 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 359
E04 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 359
SO3 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 360
************************************** *********************
EO7 TGTTCTCGGTGCAGCGTCCCGACACGCGGCCAGCGCATCGGCCCTTAGGACCCATCGAGC 416
EO1 TGTTCTCGGTGCAGCGTCCCGACACGCGGCCAGCGCATCGGCCCTTAGGACCCATCGAGC 418
EO8 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 420
EO2 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 419
E03 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 420
E06 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 418
S02 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 417
S01 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 418
E05 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 419
N02 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 419
C01 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCTGAGGACCCATCGAGC 419
N01 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 419
E04 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 419
SO3 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 420
********************* ********************** **************
EO7 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 461
EO1 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCAGGA 463
สำนกัหอ
สมุดกลาง
70
EO8 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 465
EO2 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 464
E03 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 465
E06 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 463
S02 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 462
S01 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 463
E05 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 464
N02 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 464
C01 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 464
N01 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 464
E04 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 464
SO3 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 465
***************************************** ***
14
14 GenBank
GQ . GI: 192-657 bp (Neamsuvan,O., Veldkamp,J.F.,
and Seelanan,T, ) 53 465 11.40%
139
14 80.35% 32
14 18.45%
2 14 1.16%
transition 38.57%
transversion 61.43% transition
C T 26 transversion
G C 17 C
G
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71
E01, E07 E08
4.4 4.3
4.4 rRNA gene
GenBank GQ . GI: - bp
(Neamsuvan,O., Veldkamp,J.F., and Seelanan,T, )
(+)
สำนกัหอ
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72
4.3 rRNA gene
14
C01 N01 N02 E01 E02 E03 E04 E05 E06 E07 E08 S01 S02 S03
465 465 465 465 465 465 465 465 465 465 465 465 465 465
A G 2 1 1 4
G A 6 1 1 6 2 1 1 18
T C 1 1 1 1 2 6
C T 3 1 1 5 2 1 1 1 1 4 2 1 1 2 26
% transition 100.0 33.33 66.67 51.85 31.25 16.67 25.0 33.33 40.0 31.25 28.57 16.67 66.67 42.86 38.57
A T 1 1 2
A C 3 5 1 9
G T 4 1 1 4 2 2 14
G C 2 2 1 1 1 3 4 1 2 17
C A 1 2 1 1 5 1 11
C G 1 3 2 1 1 1 1 1 3 14
T A 1 2 1 1 6
T G 1 2 2 1 2 1 1 1 1 1 1 13
%transversion 0 66.67 33.33 48.15 68.75 83.33 75.0 66.67 60.0 68.75 71.43 83.33 33.33 57.14 61.43
1 1
2 2 2 3 2 1 2 2 3 5 1 2 3 2 32
Phylogenetic tree MEGA 5.1
14
Sequence 4.4
สำนกัหอ
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73
4.5 Phylogenetic tree 14
MEGA 5.1
สำนกัหอ
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74
5
Chrysopogon aciculatus (Retz.) Trin.
14 14
DNA
GF- Plant DNA Extraction Kit DNA
PCR dye terminator sequencing
SPSS for Windows V 8.0
One-way ANOVA
1.
1.1
14 ,
, (p<0.05)
E07, E08 E02 E08 ,
E07 E
1.2 ribosomal RNA gene
primer ribosomal RNA gene
Chrysopogon aciculatus (Retz.) Trin. Genbank
GQ 63 . GI: 99 3 99 693 bp (Neamsuvan,O.,
สำนกัหอ
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75
Veldkamp,J.F., and Seelanan,T, 200 ) primer DNA
PCR 14 512 bp
DNA dye terminator sequencing
1.
14
Genbank GQ 63 . GI: 99 3 99 (Neamsuvan,O., Veldkamp,J.F., and Seelanan,T,
9) 1 2 – 2 8 bp
Phylogenetic tree
2.
14 , ,
(p< . )
E , E
E C01, S01, S02, S0 , E01,
E0 , E04, E05, E0 , N01 N02
primer primer primer
Chrysopogon aciculatus (Retz.) Trin. ribosomal RNA gene
DNA PCR DNA
ribosomal RNA gene 14
14
ribosomal RNA gene
สำนกัหอ
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76
ribosomal RNA gene
Kathleen et al,
sand live oak (Quercus geminate)
sand live oak
DNA microsatellites
3.
1.
2.
. bard
4. Blind test
สำนกัหอ
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77
. (2551) “ -
Oryza japonica L. Oryza
indica L.” 2, 2: 9-27
. (2530). . : .
. (2544). . :
.
. (2552). . :
.
. (2537). .6 5. : .
. (2535). . : .
. (2548). . :
.
. (2553). . :
.
. (2545). . 2. :
.
. (2552). : . :
.
.(2555). 5 .
17 . http://statistic.police.go.th/dn_main.htm
. (2555). . 17 .
http://web3.dnp.go.th/botany/detail.aspx?words=%CB%AD%E9%D2%E0%A8%E9
%D2%AA%D9%E9&typeword=group
สำนกัหอ
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78
Craft, Kathleen J, Jeffrey D. Owens, and Mary V. Ashley.(2006). “Application of plant DNA
markers in forensic botany: Genetic comparison of Quercus evidence leaves to crime
scene trees using microsatellites.” Forensic Science International 165 (May): 64-70
Lian Lay Hoonh, and Koh Chong Lek.(2005). “Genetic polymorphisms in mitochondrial DNA
hypervariable regions I, II and III of the Malaysian population.” Asia Pacific Jaurnal
of Molecular Biology and Biotedmology 13, 2 (October): 79-85
Li-Chin Tsai, and others. (2006). “Species identification using sequences of the trnL intron and
the trnL-trnF IGS of chloroplast genome among popular plants in Taiwan.” Forensic
Science International 164 (February): 193-200
Bradoo, Sapna D., and Anja-Katrin Bielinsky. (2010). DNA Replication and Checkpoint
Control in S Phase. Accessed April 17. Available
http://www.nature.com/scitable/topicpage/dna-replication-and-checkpoint-control-in-
s-14202419
Department of Chemistry, University of Warwick. (2009). PCR . Accessed April 17. Available
http://www2.warwick.ac.uk/fac/sci/chemistry/research/arodger/arodgergroup/people/
msrhab/ research/pcr/
Encyclopædia Britannica, Inc.. (1998). double helix: DNA structure. Accessed April 17.
Available http://kids.britannica.com/elementary/art-107613/The-structure-of-DNA-is-
a-double-helix
Hernandez, Jennifer. (2010). Profile of hot start PCR reaction. Accessed April 17. Available
http://jenniferhernandez.wordpress.com/
International Union for Conservation of Nature. (2006). Plants. Accessed April 17. Available
http://www.iucnredlist.org/
Julia Scher. (2012). Forestry Images. Accessed April 17. Available
http://www.forestryimages.org/browse/detail.cfm?imgnum=5376365
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Kenneth Todar. (2012). Todar's Online Textbook of Bacteriology . Accessed April 17.
Available http://textbookofbacteriology.net/themicrobialworld/origins.html
Mammalian Genetics University of Florida.(2011). DNA Sequencing Facilities Center for
Mammalian Genetics University of Florida. Accessed April 19. Available
http://www.dna-sequencing-service.com/dna-sequencing/dna-sequencing/
Martin F. Wojciechowski. (2006). Structure of plant chloroplast genome . Accessed April 17.
Available http://tolweb.org/notes/?note_id=4148
National Human Genome Research Institute (NHGRI) Glossary of Genetic Terms. (1999).
Nucleotide. Accessed April 17. Available from
http://www.ncbi.nlm.nih.gov/Class/MLACourse/
Original8Hour/Genetics/nucleotide.html
Neamsuvan, O., Veldkamp, J.F., and Seelanan, T. (2012). Chrysopogon aciculatus voucher O.
Neamsuvan 161 (PSU) 18S ribosomal RNA gene, partial sequence; internal
transcribed spacer1, 5.8S ribosomal RNA gene, and internal transcribed spacer2,
complete sequence; and 28S ribosomal RNA gene, partial sequence. Accessed
February 15. Avaibable from http://www.ncbi.nlm.nih.gov/nuccore/GQ856352.1
Nelson, David L., and Michael M. Cox. (2000). Nucleotides and Nucleic Acids. Accessed April
17. Available from http://www.biologyreference.com/Mo-Nu/Nucleotides.html#b
Peter Koeppel. (2012). Nucleotides – building blocks of life. Accessed April 17. Available from
http://www.nucleotidenutrition.com/nucell-im-featured-in-the-article-nucleotides-the-
building-blocks-of-life/
Pharmacia Biotech Inc.(2012). DNA Sequencing. Accessed April 17. Available
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html
Stephen L. Dellaporta. (2006). Mitochondrial genome of Trichoplax adhaerens supports
Placozoa as the basal lower metazoan phylum. Accessed April 17. Available
http://www.pnas.org/content/103/23/8751/F1.expansion.html
สำนกัหอ
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(2002). DNA as the Genetic Material. Accessed April 17. Available
http://home.earthlink.net/~dayvdanls/dna1.htm
(2012).Warm Season Turfgrasses. Accessed April 17. Available
http://www.toyogreen.com/landscape/Turfing.html
(2013). NUCLEIC ACIDS A: THE STRUCTURE OF DNA. Accessed April 17. Available
http://employees.csbsju.edu/hjakubowski/classes/ch331/dna/oldnastructure.html
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81
สำนกัหอ
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82
.
สำนกัหอ
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83
(Gel Electrophoresis)
1. 0.5M EDTA
EDTA 9.306 deio H2O 30 1N NaCl
pH pH 8 1N NaCl
50 deio H2O
2. 50X TAE Buffer
Tris 24.2 Glacial acetic acid 5.71
100 0.5M EDTA
10
3. 1X TAE Buffer
50X TAE Buffer 10 490
4. Agarose gel
agarose powder 0.4 1X TAE Buffer 40
สำนกัหอ
สมุดกลาง
84
.
สำนกัหอ
สมุดกลาง
85
1.
Oneway
2.
Oneway
3.
Oneway
สำนกัหอ
สมุดกลาง
86
4.
Oneway
5.
Oneway
6.
Oneway
สำนกัหอ
สมุดกลาง
87
.
primer
สำนกัหอ
สมุดกลาง
88
Chrysopogon aciculatus ribosomal RNA(rRNA)
gene
สำนกัหอ
สมุดกลาง
89
primer primer 3 Genbank:
GQ856352.1
สำนกัหอ
สมุดกลาง
90
สำนกัหอ
สมุดกลาง
91
.
สำนกัหอ
สมุดกลาง
92
( . .)
( . .)
( . .) ( . .)
( . .) ( . .)
S01
0.55
0.55
0.50
0.05
0.05
0.05
4.00
4.00
3.60
0.30
0.45
0.50
6.70
4.70
5.70
23.10
24.50
17.20
S02
0.50
0.60
0.60
0.05
0.05
0.05
3.80
4.00
3.60
0.40
0.60
0.40
4.80
4.60
3.20
29.00
26.30
18.20
S03
0.85
1.00
1.00
0.10
0.10
0.10
4.10
7.55
6.95
0.50
0.60
0.60
9.00
7.60
8.10
50.00
48.60
48.90
E01
0.50
0.40
0.50
0.05
0.05
0.05
4.30
5.40
4.90
0.45
0.60
0.50
4.30
3.90
4.00
19.90
29.00
20.60
E02
0.70
0.60
0.70
0.05
0.05
0.05
2.70
2.65
3.05
0.55
0.40
0.60
5.90
7.20
6.60
40.00
46.70
59.50
E03
0.75
0.60
0.80
0.05
0.05
0.05
5.20
3.40
4.60
0.30
0.45
0.50
5.20
4.20
6.10
35.00
28.20
25.20
E04
0.90
0.95
0.85
0.15
0.20
0.15
6.10
4.50
5.10
0.60
0.45
0.45
4.90
5.30
7.20
25.20
30.40
40.40
สำนกัหอ
สมุดกลาง
93
( )
( . .)
( . .)
( . .) ( . .)
( . .) ( . .)
E05
0.95
1.00
0.90
0.10
0.15
0.15
8.50
20.40
4.85
0.60
0.55
0.45
7.30
8.85
8.00
54.20
73.00
55.10
E06
1.10
1.00
1.20
0.10
0.10
0.10
6.65
6.40
7.65
0.70
0.65
0.70
7.50
9.80
6.90
45.40
52.00
63.70
E07
0.80
0.80
0.90
0.10
0.10
0.10
4.00
4.40
3.70
0.50
0.60
0.40
7.60
7.40
7.00
43.60
39.40
42.80
E08
0.90
0.85
0.80
0.10
0.10
0.10
5.20
3.15
4.60
0.45
0.50
0.45
6.05
8.90
6.30
38.50
67.10
37.70
C01
0.80
0.90
1.00
0.10
0.10
0.10
13.50
14.20
15.50
0.35
0.40
0.45
11.30
9.20
9.00
59.70
43.50
40.60
N01
1.00
0.85
0.70
0.10
0.10
0.15
8.40
20.00
12.90
0.90
0.70
0.60
9.40
8.10
9.80
55.30
69.80
81.50
N02
0.70
0.60
0.80
0.10
0.10
0.10
3.60
2.70
3.50
0.50
0.50
0.50
6.70
6.80
7.10
38.10
25.60
37.90
สำนกัหอ
สมุดกลาง
94
-
1060
74000
. . 2549
. . 2553
. . 2549-2552
. . 2553
. . 2554 –
สำนกัหอ
สมุดกลาง