ĐÁNH GIÁ ĐA DẠNG DI TRUYỀN CỦA GIỐNG LAN RỪNG Dendrobium THU THẬP TẠI THÀNH PHỐ HỒ CHÍ MINH VÀ RỪNG NAM CÁT TIÊN BẰNG KỸ THUẬT RAPD pdf
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Dendrobium T
H
2007
Dendrobium T
H
2007
TS.
CN.
iii
:
-
-
-
:
-
- TS.
-
,
.
TP. HCM
,
Nai
.
, ,
.
iv
,
.
, 9/2007.
Dendrobium
: TS.
, TS. , CN.
.
:
,
.
,
,
Dendrobium. Dendrobium
, ,
. ,
Dendrobium,
,
,
.
h
cao.
,
.
,
10
Dendrobium
c
Version 2.1 Winboot.
. 6
: OPAC10, OPAH13, 0PB01, OPB08, S1384,
U693
.
, x
10
Dendrobium.
,
h 5 0.39 0.79.
v
SUMMARY
Genetic diversity analysis of wild Dendrobium stem at Ho Chi Minh city
and Nam Cat Tien forest by RAPD
Supervisor: Tran Thi Dung
1
, PhD; Vo Thai Dan
2
, PhD; Luu Phuc Loi
1
, BSc
Student: Truong Minh Dung
1
1
Department of Biotechnology, Nong Lam university, Ho Chi Minh city
2
Faculty of Agriculture, Nong Lam university, Ho Chi Minh city
To analysis the genetic diversity of wild Dendrobium orchid, six decamers were
used for 10 wild Dendrobium species, collected from Ho Chi Minh city and Nam
Cat Tien forest (Dong Nai province). Collecting 10 species from Ho Chi Minh city
and Nam Cat Tien forest (Dong Nai province), optimizing of protocols for DNA
extraction and purification, constructing of protocol for RAPD, analyzing the
genetic of 10 wild Dendrobium species by RAPD and constructing the dendrogram
by NTSYSpc 2.1 software, Winboot program are the objective of the study.
Methods
- To identify 10 wild Dendrobium species with genotype.
- Extraction of total DNA from fresh tissue of 10 wild Dendrobium species.
- Six random primer: OPAC10, OPAH13, OPB01, OPB08, S1384, U693 were
used to analysis of genetic diversity.
- Following RAPD reactions, dendrogram were constructed by NTSYSpc 2.1
and Winboot program.
Conclusion
Six decamers are enough to compare the identity of all samples. We could use
those primers to establish the methodology to perform genetic comparisons in
Vietnam. Besides, the genetic diversity among 10 wild Dendrobium species were
established to support the breeder overcome difficulties in plant salection.
vi
Trang
iii
iv
SUMMARY v
vi
ix
x
x
xi
1.
1
1.1. 2
1.2.
ung
2
2.
3
Dendrobium 3
3
Dendrobium 5
6
8
11
11
14
14
2.3.2.2. Primer 14
2.3.2.3. dNTP 16
2.3.2.4. MgCl2 17
2.3.2.5. Taq 17
vii
2.3.5.6. Buffer 17
18
18
18
18
19
19
2.5.
Winboot 26
2.5.1. NTSYSpc version 2.1 26
2.5.2. Winboot 28
2.6 29
2.6.1. 29
2.6.2. nh h 31
3.
32
32
32
3.1.2 32
32
Dendrobium 32
3.2.2. T- 34
34
36
36
36
- 45
4.1.
Dendrobium
45
viii
4.2.
48
4.3.
53
4.4.
10
Dendrobium
d 54
4.5.
10
Dendrobium
b 2.1 Winboot 60
- 69
5.1.
69
5.2.
70
71
ix
AFLP: Amplified Fragment Length Polymorphism
bp: base pair
CTAB: Cetyl Trimethyl Ammonium Bromide
DNA: Deoxyribonucleic acid
dNTP: Deoxyribonucleotide triphosphate
EB: extraction buffer
EDTA: Ethylene Diamine Tetra acetic Acid
mM: milimolar (milimol/lite)
NCT:
OD: Optical density
PCR: Polymerase Chain Reaction
pmol: Picomol
RAPD: Random Amplified Polymorphic DNA
RFLP: Restriction Fragment Length Polymorphism
RNA: Ribonucleic acid
Rnase: Ribonuclease
SSR: Simple Sequence Repeat
Ta : Annealing t
TAE: Tris Acetate EDTA
TE: Tris EDTA
TP. HCM:
UPGMA: Unweighted Pair Group Method with Arithmetic
VNTR: variable number tandem repeat
x
Trang
11
34
37
40
42
42
4.1.
Dendrobium 46
4.2.
Dendrobium 47
B
50
4.4.
(ng/) 51
53
53
55
Dendrobium thu
62
Trang
3 qu 50
51
xi
Trang
Dendrobium 5
13
21
23
25
. 26
2.7.
2.10m 27
2.8.
Winboot 29
Dendrobium 33
4.1.
Dendrobium 45
2 49
3 49
4 50
5.
Dendrobium 54
4.6.
Dendrobium 55
7.
Dendrobium
. 56
8.
Dendrobium h. 56
9.
Dendrobium
. 57
.
Dendrobium 57
11.
Dendrobium
. 58
xii
2.
Dendrobium
. 58
3.
Dendrobium
. 59
4.
Dendrobium . 59
5.
Dendrobium
. 60
6.
Dendrobium
. 60
7.
g Dendrobium
. 61
8.
n Dendrobium
Nam
63
4.19.
Dendrobium . 64
4.20. 3
Dendrobium . 65
1
1.1.
T H , Nam
C Tin c nhiu iu kin t nhin th vic ph trin nhiu
,
Dendrobium. Lan Dendrobium
.
Dendrobium.
, ,
m
Dendrobium
. ,
lan
Dendrobium
lan Dendrobium .
n,
isozyme hay ch
(RFLP, AFLP, RAPD, SSR). T v
Dendrobium
.
2
,
:
ng di truyn ca ging lan rng Dendrobium thu th
H
.
t
Nam T H
1.2.
y
Dendrobium,
.
-
Dendrobium
.
-
.
-
.
-
Dendrobium
primer RAPD.
-
NTSYSpc version 2.1 .
-
.
-
.
- RAPD ,
.
- Dendrobium
.
- M Dendrobium
Winboot.
3
2.1. Dendrobium
Dendrobium Epidendreae Epidendroideae
Orchidaceae.
Lan Dendrobium
Dendrobium
.
Dendrobium
,
, ,
.
0
C- 33
0
C
chung, T
Dendrobium
2.1.1.
,
(photosynthese).
, ,
.
4
Dendrobium .
: h
g
Hoa: h
nh
nh
q- Q
-
5
2.1.2. an Dendrobium
Dendrobium
6
Dendrobium
Bolbidium
Callista
Apoum
Strongyle
Gastridium
Conostalix
Rhopalanthe
Formosae
Pedilonum
Dendrobium
Breviflores
Dischophyllum
Stachyobium
Callista
Dendrobium.
2.1.3.
D. anosmum
10cm -
-
D. chrysotoxum -
- - -
15cm -
7
D. densiflorum ( )cm -
- 4, thon,
b
D. farmeri
cao 20cm - -
D. fimbriatum ng
cm -
cm -
inh
, Kon Tu
D. heterocarpum
20cm - cm - 13cm
.
D. primulinum (Long Tu) ;
cm - 10
v
D. lindleyi : cao 3cm - 4cm, to 1.5cm,
cm - 30cm; hoa
cm -
8
D. thyrsiflorum
- 3cm - -
m,
D. moschatum
cm - - 9;
cm -
2.2.
(Desoxyribonuclease-DN-RNase).
m Tris HCl 1M (pH 7.5), NaCl 5M, EDTA 0.5M, sodium dodecyl
9
protein.
polysaccharide
Mc
, m
-
ethanol:
- trong isopropanol
: : 1. ng
ta n ch a trong
isopropanol.
10
nn lm. Sau
, cn ta ph u vt
n dnh li.
260 .
260nm (OD
260nm
- Optical Density
260nm
260nm
280nm
260nm
/OD
280nm
- 2.0.
11
Log u = Log u
0
K
r
*C
0
r
2.1.
% agarose
0.6 - 0.8
7 20
0.9 - 1.2
0.5 7
1.2 - 1.5
0.2 0.5
2.3. Gi
(Polymerase Chain Reaction -
,
1993
E. coli hay .
2.3.1.
PCR, h
, DN
DNA in vitro.
primer (primer
12
s i tip
nhau, mm 3 :
: - denaturation
-
0
C - 95
0
- hybridization
0
C - 70
0
- elongation
0
(polyme
36
n
13
(
