Ribosomal RNA ° Chrysopogon Aciculatus (Retz.) Trin : µ µª·· ·ª· µ r ¥µ«µ Îµ Îµ ´ ¨Îµ ´·ª ¨ : ¸Ã Å r/ Chrysopogon aciculatus (Retz.) Trin./ ´ µ ª ·

Ribosomal RNA Chrysopogon Aciculatus (Retz.) Trin

2557

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Ribosomal RNA Chrysopogon Aciculatus (Retz.) Trin

2557



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



“

Ribosomal RNA Chrysopogon Aciculatus (Retz.) Trin ”

……...........................................................

( . )

.......... .................... . ...........

.

....................................................

( . )

............/......................../..............

....................................................

( . )

............/......................../..............

....................................................

( . )

............/......................../..............



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

........................................



Chrysopogon aciculatus







Ribosomal RNA Chrysopogon aciculatus (Retz.) Trin

.

.

.

,

,



……………………………………………………………………………...

…………………………………………………………………………..

………………………………………………...……………………………

……………………………………………………..……………………………

………………………………………………..…………………………………….

1 …………………………………………………………..………………….……… 1

……….....………………….……….. 1

………………………………..…………………… 3

………………………..……………………………… 3

……………………...…………………………………... 4

……………………...……..………………… 4

………………………...………………………………... 5

……………………………………………………………… 5

…………………………………………………………... 6

2 ………………………………………………………… 7

………………………………………………... 7

………………………….………..……………………….. 11

(plant genome structure)………………………….. 13

…………… 18

………………….. 19

……………………………… 20

………………………………………………. 22



(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



. primer………………… 87

. ……………………………………………………… 91

……………………………………………………………………………... 94



2.1 …………….. 3 .

………………………………………………………………………….. 0 4. ………...……………………………………. 48 4. ..………………….... 55 4.3 rRNA gene

4 …………….…………………………………………………….. 7



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



4.3 DNA Agarose gel……………………………………………………………… 65 4.4 rRNA gene ……………….. 7 4.5 Phylogenetic tree 4 …. 73



1

1

1.

10 . . 2545 – 2554

45,402 . . 2545 30,130 . . 2554 5

, , ,

( , 2555)

, , ,

(crown)

( , 2535)

, ,



2

10,000 650


,

, 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)



3

DNA DNA primer

Chrysopogon aciculatus (Retz.)

Trin.

2.

2.1 Chrysopogon aciculatus (Retz.)

Trin. , ,

2.2


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



4

4.

4.


, , 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



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



6

beardgrass, Grass seed, Mackie’s pest, Needle grass, Pilipiliula

8.

8.1

8.2 chloroplast DNA

DNA sequencing



7

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



8

(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



9

(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)



10

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



11

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



12

(nucleoprotein) 2

1. (histone protein)

2. (nonhistone protein)

transcription factors

(gene) 3%

30,000-50,000



13

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)



14

(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



15

(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)



16

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



17

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



18

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



19

(uniparental inheritance)

(haploid)

(haplotype)

(angiosperm)

75% 25%

(gymnosperm) Pinaceae Taxodiaceae

3 4-5

Pinaceae

( , 2552)



20

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)



21

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)



22

(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



23

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



24

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



25

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



26

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

- -



27

(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)



28

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



29

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)



30

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/



31

(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)



32

(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 )



33

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)



34

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/



35

: 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 .



36

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



37

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)



38

( ,

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)



39

sand live oak DNA

microsatellites

4 2.06 x 10 -6 DNA

PCR DNA



40

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



41

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)



42

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)



43

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



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



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



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



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



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



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



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



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



52

( ) ( ) ( )

( ) ( ) ( )

4.1

( ) S01 ( ) S02

( ) S03 ( ) E01

( ) E02 ( ) E03



53

( ) ( ) ( )

( ) ( ) ( )

.

( ) E04 ( ) E05

( ) E06 ( ) E07

( ) E08 ( ) C01



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



55

4.2

S01 -

-

-

-

-

-

-

-

S -

-

-

-

-

-

-

-

S -

-

-

-

-

-

-

-



56

4.2 ( )

E01 -

-

-

-

-

-

-

-

E02 -

-

-

-

-

-

-

-

E -

-

-

-

-

-

-

-



57

4.2 ( )

E04 -

-

-

-

-

-

-

-

E05 -

-

-

-

-

-

-

-

E -

-

-

-

-

-

-

-

-



58

4.2 ( )

E07 -

-

-

-

-

-

-

-

E08 -

-

-

-

-

-

-

-

C01 -

-

-

-

-

-

-

-



59

4.2 ( )

N01 -

-

-

-

-

-

-

-

N02 -

-

-

-

-

-

-

-

( )

4.2

( ) S01



60

( )

( )

( )

4.2

( ) S02 ( ) S03

( ) E01



61

( )

( )

( )

4.2

( ) E02 ( ) E03

( ) E04



62

( )

( )

( )

4.2

( ) E05 ( ) E06

( ) E07



63

( )

( )

( )

4.2

( ) E08 ( ) C01

( ) N01



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



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



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



67


S02 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 117

S01 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 118


N02 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 119

C01 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 119

N01 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 119


SO3 ATCGACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAA 120

** *********** ****************** ************************

EO7 ATGCGATACCTGGTGTTCCTTGCCGACTACCGCGAACCATCGAGTTTTTGAACGCAAGTT 176

EO1 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 178

EO8 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 180

EO2 ATGCGATACCTGGTGTGAATTGCCGACTCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 179

E03 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 180


S02 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 177

S01 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 178


N02 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 179

C01 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 179

N01 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 179


SO3 ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTT 180

**************** **** ** * *******************************

EO7 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 236

EO1 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTAGGCGTCACGCCAAAAGACACTCC 238





68

E03 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 240


S02 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 237

S01 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 238


N02 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 239

C01 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 239

N01 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 239


SO3 GCGCCCGAGGCCTTCTGGTCGAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC 240

************************************ ***********************

EO7 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 296

EO1 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGTAGGGCGCGGTGGG 298

EO8 CAACCCACCCACGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 300

EO2 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 299

E03 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 300


S02 CAACCCACCCACGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 297

S01 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 298


N02 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 299

C01 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 299

N01 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 299

E04 CAACCCACCCACGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 299

SO3 CAACCCACCCATGGGGCAGGGACGTGGTGTTTGGCTCCCCGCGCCGCAGGGCGCGGTGGG 300

*********** ********************************** *************

EO7 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 356

EO1 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGTACGTGGTGGGCGACACCTAGT 358




69


E03 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 360


S02 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 357

S01 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 358


N02 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 359

C01 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 359

N01 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 359


SO3 CCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTGGGCGACACCTAGT 360

************************************** *********************

EO7 TGTTCTCGGTGCAGCGTCCCGACACGCGGCCAGCGCATCGGCCCTTAGGACCCATCGAGC 416

EO1 TGTTCTCGGTGCAGCGTCCCGACACGCGGCCAGCGCATCGGCCCTTAGGACCCATCGAGC 418

EO8 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 420

EO2 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 419

E03 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 420


S02 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 417

S01 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 418


N02 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 419

C01 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCTGAGGACCCATCGAGC 419

N01 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 419


SO3 TGTTCTCGGTGCAGCGTCCCGGCACGCGGCCAGCGCATCGGCCCCGAGGACCCATCGAGC 420

********************* ********************** **************

EO7 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 461

EO1 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCAGGA 463



70



E03 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 465


S02 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 462

S01 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 463


N02 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 464

C01 ACCGCAGCGCAAGCCGCTCGGACCGCGACCCCAGGTCAGTCGGGA 464

N01 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



71

E01, E07 E08

4.4 4.3

4.4 rRNA gene

GenBank GQ . GI: - bp

(Neamsuvan,O., Veldkamp,J.F., and Seelanan,T, )

(+)



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



73

4.5 Phylogenetic tree 14

MEGA 5.1



74

5


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.,



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



76

ribosomal RNA gene

Kathleen et al,

sand live oak (Quercus geminate)

sand live oak

DNA microsatellites

3.

1.

2.

. bard

4. Blind test



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



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/

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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

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International Union for Conservation of Nature. (2006). Plants. Accessed April 17. Available

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Julia Scher. (2012). Forestry Images. Accessed April 17. Available

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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

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building-blocks-of-life/

Pharmacia Biotech Inc.(2012). DNA Sequencing. Accessed April 17. Available

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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





81



82

.



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



84

.



85

1.

Oneway

2.

Oneway

3.

Oneway



86

4.

Oneway

5.

Oneway

6.

Oneway



87

.

primer



88

Chrysopogon aciculatus ribosomal RNA(rRNA)

gene



89

primer primer 3 Genbank:

GQ856352.1



90



91

.



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



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



94

-

1060

74000

. . 2549

. . 2553

. . 2549-2552

. . 2553

. . 2554 –



Documents

Ribosomal RNA ° Chrysopogon Aciculatus (Retz.) Trin : µ µª·· ·ª· µ r ¥µ«µ Îµ Îµ ´ ¨Îµ ´·ª ¨ : ¸Ã Å r/ Chrysopogon aciculatus (Retz.) Trin./ ´ µ ª ·