TRNG I HC PHNG NG

KHOA CNG NGH SINH HC V MI

TRNG

N TT NGHIP
TI: : ng dng ca ch th phn t

xc nh dng b m phc v cho chn to

ging khoai ty khng bnh mc sng
GVHD: TS. L Hng Lnh
SVTH : V Th Phng Tho
MSSV: 510301028

Mc ch v ni dung nghin cu
Chng 1: Tng quan ti liu

NI DUNG
CHNH

Chng 2: Vt liu v phng

php nghin cu
Chng 3: Kt qu v tho
lun
Kt lun v ngh

Mc ch v ni dung nghin cu
Mc ch
Xc nh c b m mang gen khng bnh
mc sng ca khoai ty bng phng php
s dng cc ch th phn t.
Ni dung
Phn tch ch th phn t a hnh gia cc
ging/dng khoai ty dng lm b m phc v
phn tch kiu gen trong qun th.

CHNG 1: TNG QUAN TI LIU

1.1. Gii thiu chung v cy khoai
ty
Cy khoai ty tn khoa hc
Solanum tuberosumL
Gii : Plantae.
B : Solanales.
H : Solanaceae.
Chi : Solanum.
Loi : S.tuberosum
Cy khoai ty l loi cy
nng ngip, trng ly c
cha tinh bt rng ri nht
th gii.

Tnh hnh sn xut cy khoai ty Vit Nam

Cy khoai c ngi Php
mang n Vit Nam t hn 100
nm trc y
Hin nay, 70% din tch khoai
ty tp trung cc tnh ng
bng Sng Hng v khoai ty
tr thnh cy trng v ng
quan trng ti y.
Vit Nam, cy khoai ty c
trng trn din tch 40.000 ha v
sn lng t 440.000 tn/nm.
Sn lng ny mi ch p ng
c 20 n 25% nhu cu tiu
dng trong nc
Tim nng ca cy khoai ty
nc ta cn rt ln.

1.2. Bnh mc sng cy khoai ty

Bnh mc sng khoai ty gy bi loi nm c tn khoa hc
Phytopthora infestans, thuc h Pythiaceae
Tc hi

Tc ly
lan v pht
trin rt
nhanh

Sau 2-3 tun

cy nhim
bnh c th
cht hon
ton

Bnh mc
sng gy
thit hi
nng sut
ln n
70%

mt trong nhng bnh nghim trng nht

trong lch s nhn loi i vi cy khoai ty

Triu chng ca bnh mc sng vi cy khoai ty

Triu chng trn l khoai ty
Nhng vt thng ban u rt nh c kch thc
t 2-10 mm v hnh thi c th khc nhau, phn b tn
thng do bnh ngy cng ln ln v to thnh
cc vng trn ng tm ca vt bnh trn l.

Triu chng trn cnh, thn

Thn cy b tht li
L phn pha trn thn b ho
Vt bnh c mu sc t sng n ti
to nn cc vng trn trn thn
Khi b nng thn cy d dng b gy.

Triu chng trn c

C b bnh c vt bnh hnh a mu sc nu en

to nn hoa vn m trn c
Mu sc c b bin mu.
C b nhim bnh tr nn cng, kh v chc li.

ng dng ch th phn t trong chn to ging

khoai ty khng bnh mc sng.
Cc ch th phn t nh AFLP, RAPD, SSR c s
dng rng ri trong cc nghin cu chn ging v di
truyn ca khoai ty
S dng ch th SSR cho php ti hin kt qu tt hn
v mc a hnh cao hn so vi ch th RAPD
Hin nay, mc biu hin a hnh trong phn tch
AFLP cao hn so vi phn tch SSR. Tuy nhin, SSR
l ch th ng tri, vic thc hin phn tch n gin
hn, khng cn cc thit b phc tp nh phn tch
AFLP.
Nh vy s dng ch th SSR l u vit nht

CHNG 2: VT LIU V PHNG PHP NGHIN

CU

1. Vt liu nghin cu
. Vt liu nghin cu gm 10 ging nhp ni
(Ging s 47; Ging s 106; Ging s 113;
Ging s 115; Ging s 116; Ging s 119;
Ging s 124; Ging s 129; Ging s 140;
Ging SOLARA) v 2 ging KT3, PO3 ang
c trng i tr a phng.
.8 ch th phn t SSR c s dng xc nh ch
th a hnh gia vt liu b m trong nghin cu

CC CP MI SSR DNG TRONG NGHIN CU KHOAI TY

S TT

Tn mi

STI 0006

LP6

Pota6

Pota 16

Pota 48

Pota 40

Pota 43

Pota 45

Trnh t
F: ctttagtccttggcagagctt
R: cgggctgattcttcttcatc
F:tgcctactgcccaaaacat
R:actggctgggaagcatacac
F: tattcccccttcctactcaa
R: tcttccacattcctaacctg
F: ttctgatttcatgcatgtttcc
R: atgtgtggtctacaaaaagggg
F: ttgcgtgaagcagccgtaaa
R: gcccagtaagtaaaacattg
F: ttcaaagaaatcacaaaatc
R: tcaagaccccaaaactaact
F: aacattacaacacattagca
R: aacttatctgaaactctcgt
F: attctccgaaagtatgtct
R: tgggatgagttggtgtgtc

Kch thc on gen

nhn bn, bp

Nhit gn mi
thc t, oC

200-250

56

128-166

57

202

55

236

60

110

54

145

51

204

52

450

52

2.2. Phng php nghin cu

1. Tch DNA tng s bng
phng php CTAB
2.Xc nh nng v tinh
sch ca DNA tng s bng
phng php quang ph k
3. Kim tra nguyn vn ca
DNA tng s bng phng
php in di trn gel agarose
4.Tin hnh phn ng PCR
5. Phn tch kt qu PCR bng
k thut in di trn gel
polyacrylamide

CHNG 3: KT QU V THO LUN

1.Kt qu kim tra DNA tng s tch chit theo phng
php CTAB trn gel agarose 0,8%

.Cc mu DNA khng b t gy, vic loi b ARN bng

RNase tin hnh kh tt th hin cc bng in di gn, r.
Nhng mu DNA ny iu kin s dng cho th nghim
sinh hc phn t tip theo.

2. Nng v ch s tinh sch ca DNA tng s

Mu

Nng (ng/l)

Ch s tinh sch
(A)260/(A)280

47

560,0

1,80

106

908,7

1,90

113

1096,1

1,92

115

1269,6

1,92

116

849,5

1,91

119

731,7

1,88

124

832,1

1,90

129

1114,8

1,92

140

782,0

1,80

SO

699,1

1,85

KT3

977,1

1,90

PO3

1112,1

1,85

Kt qu tch chit DNA cho thy tt c cc mu DNA

u c ch s tinh sch nm trong khong 1,8 2,0 nh vy
100% s mu DNA tinh sch,

3. Kt qu thu thp pht trin ch th v tm nhit

gn mi c hiu ca phn ng PCR
Kt qu chng ti xc nh v chn lc c 8 ch
th phn t SSR s dng trong nghin cu xc nh
ch th a hnh gia dng b m.
xc nh c nhit gn mi c hiu cho 8
cp mi nghin cu

4. Kt qu kho st a hnh gia cc ging cho v

nhn QTL/gen khng bnh mc sng
a hnh gia hai ging/dng khoai ty c th c pht
hin bng chiu di khc nhau ca cc on lp li c
khuych i bi phn ng PCR khi s dng cng mt
cp mi SSR.
Trong nghin cu di truyn tnh khng bnh mc sng
khoai ty chng ti s dng 8 ch th phn t trn cc
nhim sc th khc nhau xc nh ch th phn t a
hnh gia 12 dng/ging b m khc nhau, phc v
cng tc lai to chn lc ging khng

CH TH PHN T STI0006
Kt qu a hnh r rt gia cc ging
Sn phm gen nhn bn c kch thc nm trong khong 200-250bp. S
lng allen xut hin cc ging nghin cu nm trong khong t 1 n
4.
S Allen xut hin nhiu nht l cc Ging s 47, Ging s 115, Ging
s 119, Ging s 124 v Ging KT3 vi 4 allen. S Allen xut hin t nht
l Ging PO3 vi 1 allen. Cc ging cn li c t 1 n 3 allen

 Chia 12 ging nghin cu thnh 5 nhm phn bit:

Nhm 1 gm 5 ging khng a hnh vi nhau l:
Ging s 47; Ging s 115; Ging s 119; Ging s
124; v Ging KT3.
Nhm 2 gm 2 ging khng a hnh vi nhau l:
Ging s 116 v Ging s 129.
Nhm 3 gm 2 ging khng a hnh vi nhau l:
Ging s 140 v Ging SO.
Nhm 4 gm 2 ging khng a hnh vi nhau l:
Ging s 106 v Ging s 113.
Nhm 5 ch c Ging PO3. Nh vy, Ging PO3 a
hnh vi tt c cc ging cn li

CH TH PHN T LP6
Gen nhn bn c kch thc khong 128-166 bp, s bng gen
phn tch nhiu (t 3 n 10 bng gen). S allen nhiu nht l
Ging s 140 vi 10 allen
Ch c 2 Ging s 116 v Ging s 129 khng a hnh vi
nhau. Tt c ging cn li u a hnh vi nhau v a hnh
vi Ging s 116 v Ging s 129.

CH TH PHN T POTA 16

Gen c nhn bn c kch thc 236 bp v s allen

ca tt c cc ging u bng 2

 Kt qu phn thnh 3 nhm ring bit

Nhm 1 gm 5 ging khng a hnh vi nhau l:
Ging s 106, Ging s 119, Ging s 124, Ging s
129, v Ging s 140.
Nhm 2 gm 6 ging khng a hnh vi nhau l:
Ging s 47, Ging s 113, Ging s 115, Ging s
116, Ging SO, v Ging KT3.
Nhm 3 ch c 1 ging PO3. Nh vy ging PO3 a
hnh vi tt c ging cn li.

CH TH PHN T POTA 6

Sn phm PCR c kch thc 202 bp.

S Allen cc ging thay i trong khong t 2 n 8. Trong
, Ging s 47 v Ging s 106 c s allen nhiu nht vi 8
allen.
Ging c s allen t nht l Ging s 113, Ging s 119,
Ging s 124, Ging SO v Ging PO3 vi 2 allen

 12 ging nghin cu c th phn ra thnh 5 nhm khc

nhau v c tnh di truyn.
Nhm 1 gm Ging s 47 v Ging s 106 khng a hnh
vi nhau.
Nhm 2 gm 5 ging khng a hnh vi nhau l Ging
s 113, Ging s 119, Ging s124, Ging SO, v Ging
PO3.
Nhm 3 gm Ging s 115 v Ging s 116 khng a
hnh vi nhau.
Nhm 4 gm Ging s 140 v Ging KT3 khng a hnh
vi nhau.
Nhm 5 ch c mt Ging s 129. Nh vy, Ging s 129
a hnh vi tt c ging cn li

CH TH PHN T POTA 48

Kt qu gen nhn bn vi ch th POTA48 c kch

thc 110 bp v s allen thay i t 1 n 2. Ging
s 116 v Ging SO c s allen nhiu nht vi 2
allen

 Kt qu phn thnh ba nhm ring bit.

Nhm 1 gm 9 ging khng a hnh vi nhau l Ging s
47, Ging s 113, Ging s 115, Ging s 119, Ging s
124, Ging s 129, Ging s 140, Ging KT3, v ging
PO3.
Nhm 2 gm 2 ging l Ging s 116 v Ging SO.
Nhm 3 ch c Ging s 106. Nh vy, Ging s 106 cho
kt qu a hnh vi tt c ging cn li

S alen a hnh v gi tr PIC ca cc ch th phn t

SSR phn tch vi 12 ging khoai ty
Tng s

Ch th

S allen a

SSR

hnh

STI0006

37

0.74

POTA6

46

0.82

POTA16

24

0.64

LP6

14

73

0.91

POTA48

14

0.36

Tng

32

194

Trung bnh

6,4

38,8

STT

allen a
hnh

Ch s a
hnh PIC

0,694

Gi tr ch s a hnh PIC bng 0 ti v tr

locus ch c 1 allen n hnh. Cc ch th
SSR c gi tr PIC ln hn hoc bng 0.5
s cho s phn bit cao v t l a hnh ca
ch th . Nh vy, qua bng trn ta nhn
thy c 4/5 ch th c gi tr PIC > 0.5.
iu ny khng nh rng, 80% s ch th
s dng trong nghin cu c s khc bit
ln v t l a hnh.

KT LUN
Tch chit DNA khoai ty bng phng php
CTAB cho hiu qu cao, tch chit c 12
mu DNA khoai ty tinh sch, cht lng
tt.
S dng 8 ch th phn t nhng ch c 5 ch th
phn t s dng nhn cc on DNA trong
12 ging khoai ty u cho a hnh cao v c
th phn bit s khc bit gia cc ging
Chn c 2 ging khoai ty l ging s KT3
v 116 dng lm b m phc v cho chn to
ging khoai ty khng bnh mc sng.

 NGH
Trong th nghim ny ti nghin cu c 5
ch th phn t xc nh dng b m phc
v chn to ging khoai ty khng bnh mc
sng, trong thi gian ti ti s tin hnh
nghin cu thm nhiu ch th phn t hn na
xc nh dng b m phc v chn to
ging khoai ty khng bnh mc sng.

Cm n thy c v cc bn lng nghe