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Đánh giá sự đa dạng di truyền của giống lan rừng Dendrobium thu thập

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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 Tin c nhiu iu kin t nhin th vic ph trin nhiu
    , 





  Dendrobium. Lan Dendrobium 
















 . 


 
 


  

Dendrobium.


 ,  ,
m 

 

 

 Dendrobium 










.  ,              
lan 


Dendrobium 



 
          
lan Dendrobium .
n, 



 
 isozyme hay ch 
(RFLP, AFLP, RAPD, SSR). T v



Dendrobium 

.


2






,  














 : 




ng di truyn ca ging lan rng 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      
           


 
            

                




Mc 
         , m     


-             

ethanol: 

- trong isopropanol
: : 1. ng
      ta n     ch  a trong
isopropanol.


10
 nn lm. Sau
, cn ta ph u vt
n dnh li.




     
 




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 tip
nhau, mm 3 :
: - denaturation

- 

0
C - 95
0

 - hybridization
 

0
C - 70
0


 - elongation
      
0

      
(polyme




36



n

 


13



(

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