Chng 3

PHNG PHP PHN TCH A DNG DI TRUYN

CA SINH VT
Mc tiu
- Trnh by v phn tch c khi nim v cc mc biu hin tnh a dng di truyn ca
sinh vt;
- Tip cn c phng php phn tch tnh a dng di truyn ca sinh vt mc qun
th, mc phn t (DNA v protein)
- S dng c phn mm trn my vi tnh phn tch s a dng v mi quan h di
truyn gia cc i tng nghin cu trn c s phn tch cc phn on DNA, trnh t
nucleotide ca gen, trnh t amino acid.
Ti liu hc tp
1. Chu Hong Mu (2008). Phng php phn tch di truyn hin i trong chn ging
cy trng. Nxb i hc Thi Nguyn.

Ni dung bi ging
1. KHI NIM V BIU HIN CA TNH A DNG DI TRUYN SINH VT
1.1. Khi nim
Sinh vt trn tri t biu hin s a dng, phong ph v kiu gen v kiu hnh. Ngun
gc ca tnh a dng l s phong ph v vn gen ca qun th.
a dng di truyn ca sinh vt l mt c tnh ca sinh vt. S a dng v kiu hnh do
tnh a hnh v kiu gen quy nh v c s tng tc gia kiu gen vi mi trng.
1.2. Biu hin cua tinh a dang di truyn
- a dng v kiu hnh: tnh trng hnh thi: hnh dng, mu sc ...a dng v tnh trng
s lng, v d...
- a dng v cc phn on DNA, v du:...
- a dng v trnh t nucleotide ca gen, v d...
- a dng v thnh phn protein, v d...
- a dng v trnh t amino acid.
2. PHNG PHP PHN TCH TNH A DNG KIU HNH
2.1. Phng php phn tich s a dang cc tinh trang hnh thi
- M t km theo hnh v, nh chp
2.2. Phng php phn tich s a dang cc tinh trang s lng bng NTSYS
S a dng kiu hnh l s biu hin a dng cc tnh trng ca c th trong qun th.
Da vo tr s o c ca cc tnh trng nghin cu, x l bng phn mm chuyn
dng s cho kt qu v h s a dng kiu hnh, khong cch di truyn v mi quan h
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ca cc i tng nghin cu.
- Xc nh h s sai khc bng vic thit lp bng ma trn
- Xc nh khong cch di truyn bng thit lp s hnh cy
V d: Phn tch tnh a dng v s phn ng i vi hn ca ngoi cnh
phn tch tnh a dng kiu hnh thng qua cc tnh trng th hin s phn ng i
vi hn t ngoi cnh v d trn da trn kt qu phn tch 19 tnh trng nghin cu.
Bng chng trnh NTSYS pc 2.02i nhn c h s khc nhau gia cc ging u
tng v s v mi quan h gia cc ging u tng nghin cu (Hnh 2.4).
Hnh 2.4 cho thy 22 ging u tng c phn b trong 2 nhm ln. S phn b ny
th hin mi quan h ca cc ging u tng a phng da trn s phn ng ca kiu
gen i vi hn. Trong s hnh 6, nhm I gm 18 ging: VMK, PY3, PY4, PY5,
SM1, YC, MS, QN2, MC, ML, HL, TL, K, QH, VCB, CB4, VBK, HUL; nhm II gm
4 ging: TO, LS, T84, SM3. Trong nhm I, ging CB4 c xp ring mt nhm,
nhm cn li gm 17 ging li c chia thnh cc nhm nh nhiu cp h s khc
nhau.

Nhm II

Nhm I

Hnh 2.4. S hnh cy m t mi quan h gia cc ging u tng da trn kt qu

phn tch h s khc nhau v s phn ng ca ca cc ging u tng i vi hn

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3. PHN TCH S A DNG CC PHN ON DNA (RFLP, RAPD, SSR)
3.1. C s cua tinh a dang di truyn trn c s cc phn oan DNA
- Phn tch s a dng ca sinh vt mc DNA trn c s xut hin cc phn on DNA
t RFLP, RAPD, SSR
- Cc c th khc nhau, cu trc DNA khc nhau v biu hin trnh t nucleotide v
nh vy cc on DNA cng rt khc nhau. Chnh v vy, da vo s khc nhau v cc
phn on DNA m c th nh gi c mc a dng ca sinh vt.
3.2. Phng php thng k s xut hin cc oan DNA
- Bn cht ca RFLP, RAPD, SSR
- Thng k s xut hin cc on DNA theo h nh phn (1, 0).
3.3. Quy trnh x l d liu bng NTSYS.
Phn tch s liu RAPD, SSR, RFLP c thc hin trn my vi tnh theo chng trnh
NTSYSpc Version 2.0 (Applied Biostatistisc Inc., USA., 1998). Vi mc tiu l xc nh
s ging nhau v khc nhau v cu trc DNA trong phm vi phn tch cc on; xc nh
s a hnh cc phn on DNA; tm quan h di truyn gia cc i tng nghin cu.
Nguyn tc x l cc s liu gm 3 bc sau y:
Bc 1: So snh tng cp i tng trong nghin cu bng cch tnh ton
khong cch quan h gia chng
Bc 2: Lp mt ma trn gm tt c nhng gi tr tnh ton c trc
Bc 3: Gii ma trn v biu din thnh mt biu c trng
Cc bc phn tch c thc hin nh sau:
(i) Nhp cc s liu ca bng thng k cc band in di sn phm RAPD vo bng
tnh NTEDIT.
(ii) X l kt qu bng NTSYS.
(iii) Kt qu x l s liu th hin bng h s tng ng di truyn v s
hnh cy.
Quy trnh thc hin x l s liu trn my vi tnh bng phn mm NTSYSpc Version 2.0
nh sau:
(1) Nhp d liu
a. Chn biu tng NTDIT mn hnh
b. Chn s lng ct, hng v t tn ct
c. Nhp s liu vo hng ct
d. Chn file, save file as
e. t tn file (k hiu t1)
f. Save.
(2) X l d liu
Chn menu Similarity

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 a) Chn biu tng trn mn hnh: NTSYS
b) Chn Similarity
c) Chn Simqual
d) Vo Input file (kch p)
e) Chn file t tn (t1)
f) Open (hin file trn dng input file)
g) Chn output file (kch p)
h) Chn t1 v i tn file thnh t2
i) Save
j) Chn Compute. ng Simqual.
Chn menu Clusteving
a) Chn SAHN
b) Chn Input: Kch p input: chn t2.nts, open
c) Chn Output: Kch p ly t1 v i thnh t3
d) Save
e) Chn Compute. Bng hin ra
Chn menu Graphics thit lp s hnh cy
a) Chn Tree plot
b) Chn Input file
c) Chn file t3.NTS
d) Open
e) Compute
f) Hin ra cy pht sinh
g) Chn Edit: Copy bimap
Gi kt qu phn tch h s ng dng di truyn
a) Chn file
b) Chn Edit data file
c) Chn t2.nts
d) Open c bng h s tng ng

V D: PHN TCH TNH A HNH DNA BNG RAPD (Random Amlified

Polymorphic DNA=RAPD)
RAPD
Nm 1990, Wiliam v cs pht trin k thut RAPD da trn c s ca k thut PCR.
Phn ng RAPD da trn nguyn tc ca PCR, n l phn ng nhn bn on DNA mt
cch ngu nhin bt k v tr no ca DNA nh cc mi (primers) ngu nhin c kch
thc 10bp. Thnh phn phn ng RAPD gm c DNA mu, mi (di 10 base), Taq
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polymerase (enzyme chu nhit c tch t vi khun Thermus aquaticus), bn loi
deoxirinucleotide triphotphate (dATP, dTTP, dGTP, dCTP), ion Mg2+ v dung dch m.
RAPD c t 40-45 chu k, mi chu k gm 3 giai on: (i) Giai on tch chui DNA:
DNA b bin tnh t dng si kp thnh dng si n nhit 94 0-950C trong khong
thi gian ngn; (ii) Giai on gn mi: Nhit phn ng c h thp xung 320-400C
cho php mi gn vo DNA mu v tr tng ng theo nguyn tc b sung (A - T, G -
C ); (iii) Giai on ko di: nhit 72 0C Taq polymerase hot ng ko di on
DNA t on mi v hai on DNA. Kt qu to ra 240 -245 on DNA c nhn bn.
RAPD khc PCR ch nng Mg2+ trong thnh phn ca phn ng cao hn, mi n
ngn (10bp) c th tip hp nhiu v tr trong DNA v kt qu nhn bn c s on
DNA t 2 phn on DNA tr ln. Mi on DNA c nhn c kch thc t 100
5000 bp.
RAPD cn c gi l k thut phn loi phn t, c s dng phn tch v xc
nh mi quan h thn thuc gia cc ging cy trng hay gia cc c th, phc v cho
cng tc lai to ging hoc phn loi. u im ca k thut ny l nhanh, r, n gin v
gip xc nh tnh a dng sinh hc, ngun gc v quan h di truyn ca cc ging ng
vt, thc vt, vi sinh vt. u im ca k thut RAPD l khng cn bit trnh t on
DNA cn nghin cu, quy trnh tin hnh nhanh, ch cn mt lng nh DNA khun.
Tnh a dng thu c t cc ch th RAPD c nh gi cao hn so vi k thut RFLP
v cho php pht hin c tnh a dng ngay c trong cc on cha cc trt t
nucleotide lp li. Nhc im ca ch th ny l, RAPD c tnh cht tri, do nhng
gen iu khin tnh trng no c tnh ln s kh tm thy s a hnh trn gel in di.
Cc nghin cu cho thy RAPD l mt k thut c hiu qu trong vic xc nh kiu
gen, phn tch qun th v ngun gc loi, nghin cu di truyn loi v lp bn di
truyn. K thut RAPD c s dng nhn bit v phn loi cc ging cy khc nhau,
s a dng di truyn ca cc i tng .
Da trn s xut hin hay khng xut hin ca cc phn on DNA khi in di sn phm
RAPD v c nh gi theo quy c 1 = xut hin v 0 = khng xut hin. Mt bng
gm cc gi tr 0 v 1 c thit lp t cc c th nghin cu s cho php tnh ra h s
tng ng di truyn ca cc cp i tng theo Nei v Li. Trong qu trnh thit lp mi
quan h gia cc loi hay nhm loi, Apostol v cs (1993) xy dng k thut phn
nhm thng qua cc biu RAPD (RAPDLOT).
Thc cht k thut ny gm 3 bc: (1) So snh tng cp i tng trong nghin cu
bng cch tnh ton khong cch quan h gia chng; (2) Lp mt ma trn gm tt c
nhng gi tr tnh ton c trc ; (3) Gii ma trn v biu din thnh mt biu
c trng.
Ngy nay, cc nh nghin cu thit lp c phn mm my tnh t ng v nn
biu mi quan h hay tng ng di truyn ca cc i tng nghin cu trn c
s phn ng RAPD. Sau khi nhp d liu v cc phn on DNA c nhn bn ca cc
c th, chng trnh my tnh t ng thit lp h s ng dng di truyn v lp ra biu
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 hnh cy. Biu hnh cy th hin mc gn nhau v cu trc DNA ca cc c th,
cho php nh gi c mi quan h di truyn gia cc c th c nghin cu. Chng
trnh ny cho php gim bt thi gian nghin cu v c chnh xc cao nn n l mt
phn mm c hiu qu trong vic phn tch k thut RAPD.
RAPD c s dng phn tch s a dng trong cu trc DNA, xc nh ch th phn
t RAPD v l c s cho vic thc hin phn loi phn t RAPD.
Phn tich sn phm RAPD
Sn phm RAPD c in di trn gel agarose 1,5% n 4% cng vi thang DNA chun
v chp nh di nh sng cc tm.
Cn c vo thang DNA chun v kt qu nhn bn cc on DNA, thng k cc on
DNA theo kch thc phn t v x l RAPD bng phn mm chuyn dng.
Phn tch RAPD s cung cp thng tin v tnh a dng di truyn mc DNA v mi
quan h di truyn ca cc i tng nghin cu lm c s cho chn ging.
Phn tich a dang di truyn mc DNA bng k thut RAPD
Mc tiu ca k thut RAPD l xc nh s ging nhau v khc nhau v cu trc DNA
trong phm vi s dng cc mi. Xc nh s a hnh cc phn on DNA v tm mi
quan h di truyn gia cc i tng nghin cu v lp bn cc ch th RAPD.
S dng k thut RAPD phn tch tnh a dng di truyn v thit lp quan h di
truyn ca cc i tng nghin cu, ngi ta c th tin hnh theo cc bc sau y:
(1) Chit rt DNA xc nh hm lng v tinh sch DNA
(2) Tp hp cc mi ngu nhin (10bp)
(3) Thm d iu kin ca phn ng v chu trnh nhit, nng v dung tch ca
cc thnh phn phn ng.
(4) Sng lc cc mi ngu nhin to c danh sch cc mi cho kt qu a
hnh.
(5) Thc hin phn ng RAPD vi tt c cc mi sng lc.
(6) in di kim tra sn phm RAPD
(7) Thng k cc band in di (xut hin: s 1, khng xut hin: s 0)
(8) Lp bng xc nh cc phn on RAPD c trng cho tng i tng nghin cu.
(9) Xc nh gi tr PIC, s phn on n hnh, phn on a hnh v t l %
phn on a hnh.
(10) Xc nh h s ng dng di truyn v gi tr tng quan kiu hnh (r).
(11) Thit lp s hnh cy (Dendrogram) v xc nh quan h h hng ca cc
i tng nghin cu.

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V d: S dng k thut RAPD phn tch h gen ca 12 ging u tng a phng,
sng lc vi 20 mi ngu nhin xc nh c h s ging nhau v s hnh cy m
t mi quan h di truyn ca cc ging u tng ny.

M 1 2 3 4 5 6 7 8 9 10 11 12

3Kb
2Kb
1,2Kb
0.9Kb
0.7Kb

Hnh 2.1. Hnh nh in di sn phm RAPD vi mi M6 ca 12 ging u tng a

phng min ni (1- SL1; 2-SL2; 3-SL3; 4-SL4; 5-VMK; 6-QH; 7-CB4; 8-VCB; 9-
LS; 10-HL; 11-CV; 12-TL)

T hnh nh in di ca 12 ging u tng vi 20 mi, thng k c cc phn on

DNA c nhn bn hoc khng c nhn bn. S dng chng trnh NTSYS pc xc
nh c h s ging nhau ca cc cp ging u tng v s hnh cy v mi quan
h gia cc ging u tng nghin cu.

Hnh 2.2. S hnh cy m t mi quan h di truyn ca cc ging u tng a

phng

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4. PHN TCH A DNG TRONG CU TRC GEN
S a dng trong cu trc gen th hin s khc nhau kch thc v v s lng, thnh
phn, trnh t nucleotit trong gen. S a dng trong cu trc gen l c s nh gi tnh
a dng di truyn mc phn t.
S a dng ca cc i tng nghin cu cn th hin trong trnh t nucleotide. Bng
cch so snh trnh t nucleotide ca mt gen no c th xc nh c khong cch di
truyn gia cc i tng nghin cu. Thit lp c s hnh cy th hin mi quan
h gia cc i tng.
Quy trnh phn tch s a dng trong cu trc gen:
(1) Thu thp thng tin v gen ch v thit k cp mi PCR nhn gen;
(2) Phn lp gen ch ca cc i tng nghin cu;
(3) Tch dng v gii trnh t gen;
(4) So snh trnh t gen, lp bng ma trn v h s tng ng v h s khc nhau. Thit
lp s hnh cy gia cc i tng da trn d liu v gen;
(5) So snh trnh t protein ca gen, lp bng ma trn v h s tng ng v h s khc
nhau. Thit lp s hnh cy gia cc i tng da trn d liu v protein;

V d: Phn tch a dng v gen expansin lin quan n s ko di r cy u tng

Tin hnh so snh trnh t nucleotide on m ha ca gen EXP1 ca mt s ging u
tng (DT84, SL3, SL1, LS, AF516879) vi trnh t nucleotide on m ha ca gen
EXP1 ca mt s cy trng khc (Zea may; G. lobatum v G. terneri), kt qu xc
nh h s tng ng v h s sai khc ca cc trnh t gen EXP1 (Bng 3.3).
Bng 3.3 cho thy h s tng ng v trnh t on m ha ca gen EXP1 ca 8 ging
cy trng em phn tch c mc giao ng rt ln t 47, 0% n 99,7%. H s sai khc
giao ng kh ln t 0,3% n 53%. Trong 8 trnh t on m ha gen EXP1 em phn
tch th c 5 trnh t ca cc ging u tng c mc tng ng cao dao ng t
96,2% n 99,7%, h s sai khc ca 5 ging ny dao ng t 0,3 % n 3,5%. Bng 3.3
cn cho thy trnh t on m ha ca gen EXP1 ca 5 ging u tng c h s tng
ng thp v c h s sai khc cao so vi 3 ging cy trng khc.
H s tng ng v h s sai khc ca trnh t on m ha ca gen EXP1 ca 8 ging
cy phn tch

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 S hnh cy c thit lp da trn d liu v trnh t nucleotide on m ha
ca gen EXP1 8 ging cy trng th hin Hnh 3.12.
DT84.seq
SL1.seq
AF516879.seq
SL3.seq
LS.seq
G. Lobatum.seq
G.Terneri.seq
Zea May.seq
27.9

25 20 15 10 5 0
Nucleotide Substit utions (x100)

Hnh 2.3. S hnh cy biu hin mi quan h di truyn ca 8 ging cy trng c

thit lp trn c s d liu trnh t on m ha gen EXP1

BI TP
Bi 1. Trnh by m hnh chung ca phng php phn tch s a dng di truyn ca
sinh vt bng k thut RAPD (Random Amlified Polymorphic DNA).
Bi 2. So snh s ging v khc nhau gia phn ng chui polymerase (PCR) v phn
ng random amlified polymorphic DNA (RAPD). Nu vai tr ca cc phn ng ny
trong phn tch di truyn phn t.
Bi 3. Th no l s a dng kiu hnh, s a dng trong h gen v s a dng trong cu
trc gen. Nu cc c im c bn ca k thut phn tch s a dng kiu hnh ca sinh
vt.
Bi 4. Hy gii thch s hnh cy di y v xc nh khong cch di truyn ca cc
i tng nghin cu trn c s phn tch RAPD:
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