XY DNG BN LIN KT BNG CH TH SSR V PHN TCH HIN TNG

PHN LY KHNG BNH THNG TRONG QUN TH F2

CA 2 GING LA KHANG DN 18 V IR24
Construction of linkage map by SSR marker and analysis of segregation distortion in F2
population derived from cross between 2 rice varieties Khangdan18 and IR24.
Phm Hu Anh1, Nguyn Hoi Nam1, V Huy Minh1, Nguyn Quc Trung1,2.
1
Khoa Cng ngh sinh hc, Hc vin Nng nghip Vit Nam
2
Trung tm nghin cu cy trng Vit Nam v Nht Bn (CIPR)
Tm tt:
Nghin cu s dng 93 c th ca qun th F 2 t php lai 2 ging Khang dn (KD18)
v IR24 xy dng bn lin kt xc nh cc gen hu ch ca KD18 cc nghin cu
tip theo. Kt qu kho st genome xc nh c 80 ch th SSR a hnh nm trn 12
nhim sc th (NST) gia 2 ging KD18 v IR24. S dng cng c BLAST, v tr vt l cc
locus c xc nh v bn vt l c xy dng cho 80 locus/ch th a hnh. Da trn s
phn ly ca 80 locus trong qun th F2, khong cch di truyn (cM) gia 80 locus c xc
nh bng phng trnh Kosambi v bn lin kt c xy dng s dng phn mm
MAPMAKERv.3.0 vi tng chiu di 12 NST l 2.553,7 cM. Hin tng phn ly khng bnh
thng c pht hin 4 locus nm trn NST s 3, 6, v 9 bng php kim nh 2 (P<0,05).
Kt qu nghin cu l c s tin hnh xc nh v lp bn cc gen khng su bnh v
chng chu ging KD18, ng thi l thng tin c bn v cc locus lin kt vi s cch ly
di truyn cy la.
T kha: qun th F2, bn lin kt, phn ly bt thng, ch th SSR.
1.

t vn

Lp bn lin kt da trn cc ch th ADN l mt bc rt quan trng trong xc nh

v nghin cu quy lut di truyn ca QTL/gen. Bn lin kt l c s xc nh cc nhm
lin kt v v tr ca gen trn NST. C rt nhiu bn lin kt c xy dng trn cy la
(Sasaki et al. 1994, McCouch et al. 2002). Khi phn tch s phn ly ca cc nhm lin kt,
mt s nghin cu pht hin c cc locus phn ly khng bnh thng, nh pht
hin ra cc nhm gen gii thch cho hin tng cch ly di truyn khi lai cc ging indica vi
japonica (Kinoshita, 1995; Yamamoto et al, 2007). Mt cc ch th trong bn lin kt
cng cao th cng c nhiu c hi pht hin cc phn ly khng bnh thng.
Bn lin kt u tin la c xy dng bi Nagao v Takahashi, 1963. Ngy nay,
cc bn lin kt la c cng b s dng nhiu loi ch th ADN khc nhau nh: RFLP,
AFLP, SSR, SNP trong ch th SSR c s dng ph bin nht v nhng u im ca
n nh: s lng ln, d s dng, lp li cao...
Vit Nam l nc c ngun gen thc vt a dng do c im a l, kh hu v l mt
trong nhng trung tm khi nguyn ca cy la (Molina et al. 2011). Cc ging la a
phng, ging ci tin Vit Nam c tim nng di truyn ln mang nhiu gen hu ch nh
khng su bnh v chng chu iu kin mi trng bt thun. Tuy nhin, ngun ti nguyn
ny cha c quan tm xng ng, c rt t nghin cu tin hnh trong nc lp bn lin
kt v xc nh cc QTL/gen ny phc v cho lai to, ci tin cc ging la mi. Ging
la KD18 l ging la c s dng ph bin Vit Nam trong thi gian di, y l ging
la thun, c tnh khng vi hu ht cc loi su bnh (bnh bc l, o n, ry nu) v
chng chu kh vi cc mi trng bt thun: chu lnh v chu hn tt. y l ngun gen

qu cn c khai thc ng dng trong chn to ging la mi Vit Nam ng ph vi hin

tng bin i kh hu ton cu. Da trn nhn thc c vai tr quan trng ca phng
php lp bn lin kt trn, chng ti t ra mc tiu ca nghin cu l: kho st genome
pht trin b ch th SSR a hnh t xy dng bn lin kt qun th F 2 ca 2 ging
IR24 v KD18 v phn tch cc locus phn ly khng bnh thng.
2. Vt liu v phng php
2.1

Vt liu:
Trong v ma 2015, 93 c th F 2 c trng ti khu ng rung, Trung tm Nghin
cu cy trng Vit Nam-Nht Bn, Hc vin Nng nghip Vit Nam. Bn mi tm cy ca
2 ging KD18 v IR24 c s dng lm i chng. Ht F 2 c ngm trong nc 48h
30C v 24h 30C. Cc ht thc ny mm c gieo trn khay t v c cy ra rung
sau 25 ngy vi khong cch cy l 20cm v cch hng 25cm. Mu l tng c th c thu
thp 30 ngy sau khi cy.
2.2
Phng php
Pht trin qun th F2: ging KD18 c nhiu c tnh chng chu tt c s dng lm cy
cho phn v ging IR24 mn cm vi hu ht cc bnh c chn lm cy m-nhn phn.
Trong v Ma 2012, tin hnh trng v lai KD18/IR24 bng phng php kh c ct c
tru. Ht F1 c thu v tip tc c gieo trong v Xun 2013, tin hnh cho cy F 1 t th
phn t nhin thu hoch ht F2 s dng xy dng bn lin kt.
Phng php tch chit ADN: tin hnh theo phng php ca Dellaporta et al. 1983.
La chn ch th phn t v xc nh kiu gen F2 bng ch th SSR:
Da theo kt qu kho st genome 2 ging KD18 v IR24 ca Phng th nghim Chn
ging phn t, Trung tm Nghin cu cy trng Vit Nam-Nht Bn, cc ch th SSR a hnh
c chn lc t 1906 ch th kho st. Tiu ch la chn ch th SSR l: ch th ng tri,
bng in di r nt, kch thc sn phm PCR 100-400 bp, nm ri u trn 12 NST v c
nhit gn mi 55C.
K thut PCR xc nh kiu gen cy F 2: k thut PCR c tin hnh vi bc bin tnh
o
95 C-5 pht, 30 chu k gm 95oC-30, 55oC-30, 72oC-30 v bc ko di cui 72oC-7
pht. Sn phm PCR c in di trn gel agarose 4% c b sung ethidium bromide nng
100 ng/l, tin hnh chy in di hiu in th 250 V, cng dng in 400 mA trong
thi gian 50-70 pht. Kt qu in di c quan st v phn tch trn n UV.
Da trn kch thc sn phm PCR, mu c bng ADN c trng cho KD18 m ha l
alen A, tng t vi IR24 m ha l alen B v mu d hp t khi xut hin ng thi 2 bng
ca KD18 v IR24, m ha l H.
Xy dng bn lin kt
Bn lin kt c xy dng bng vic phn tch tn s ti t hp ca 80 locus i
vi 93 c th F2 (Lander & Green, 1987) s dng phn mm MAPMAKERv.3.0. Khong
cch gia cc locus c tnh theo centiMorgan (cM), s dng phng trnh Kosambi
(Kosambi,1944). Khong cch di truyn (d) trn bn da trn tn s ti t hp (r) c
tnh ton theo cng thc ca Kosambi c loi tr cc sai s c th do hin tng trao i
cho kp:

( e4 d1)
1 1+2 r
d= ln
trong r=
4 12 r
2(e4 d + 1)
Phn tch hin tng phn ly bt thng F2
Tin hnh kim nh quy lut phn ly F2 i vi tng locus, cc locus phn ly bnh
thng s cho t l d kin l 1:2:1; locus phn ly khng bnh thng c t l khc 1:2:1. Cc
phn tch s dng kim nh 2vi mc sai s chp nhn 0,05, bc t do n-1=2. Cc locus c
hin tng phn ly khng bnh thng s c gi tr 2thc t ln hn 2l thuyt (=5,991).
3. Kt qu v tho lun
3.1. Bn vt l 80 locus gen ca 2 ging KD18 v IR24
Da trn nghin cu kho st genome 2 ging KD18 v IR24, chng ti chn lc c
80 ch th SSR cho bng ADN a hnh. Trong NST s 6 tp trung nhiu locus nht (14 ch
th) v NST s 10 c t nht vi 3 ch th (Bng 1).
Bng 1: 80 ch th/locus a hnh gia 2 ging KD18 v IR24

S dng cng c BLAST (Basic Local Alignment Search Tool) vi tham chiu l
genome ging la Oryza sativa Japonica ( IRGSP-1.0) trn trang web
http://ensembl.gramene.org, v tr vt l ca 80 locus phn b trn 12 NST c xc nh.
Bn vt l m t v tr tng i ca 80 locus c biu din trong hnh 1:

Hnh 1. Bn vt l ca 80 locus trn 12 NST la (n v Mb)

3.2. Xy dng bn lin kt
Kiu gen ca 93 cy F2 tng locus c xc nh da trn kt qu PCR s dng 80
ch th SSR. a s cc ch th cho a hnh l ch th ng tri vi mt alen c hiu, ngoi ra
c mt s t ch th c nhiu alen. Kt qu xc nh kiu gen 93 c th F 2 so vi 2 ging la
b m c minh ha hnh 1 vi ch th R9C4.

Hnh 2. Kiu gen ca 93 c th F2 c xc nh bng ch th R9C4

S liu kiu gen ca qun th F2 c phn tch bng phn mm MAPMAKERv.3.0

xc nh trt t cc locus trn NST v khong cch gia cc locus theo phng trnh
Kosambi. Kt qu xy dng c bn lin kt c tng chiu di l 2.553,7 cM. Khong
cch trung bnh gia 2 ch th l 31.9 cM. Khong cch trung bnh trn nhim sc th ngn
nht (NST s 6) l 8.6 cM v khong cch di nht (NST s 1) l 71.1 cM (Hnh 3).

Hnh 3. Bn lin kt ca qun th F2 (n v cM)

3.3. Phn tch hin tng phn ly khng bnh thng
Kim nh t l phn ly thc nghim so vi t l l thuyt (1:2:1) bng kim nh 2
vi bc t do n-1=2 v mc ngha = 0,05 pht hin mt s hin tng phn ly khng
bnh thng 80 locus. Cc locus c hin tng phn ly khng bnh thng s c gi tr 2
thc t ln hn 2 = 5,991. Kt qu c 6 locus phn ly khng bnh thng gm: R11D12 v
R25E1 trn NST s 3; R13D5 v R13E6 trn NST s 6; R15G4 v R15G1 nm trn NST s
9 (bng 2). Nguyn nhn ca hin tng ny c th do cc gen locus ny gy hin tng
bt dc giai on giao t th hoc bo t th. Do khong cch gia 2 locus trn NST s 3
v 9, c th s b kt lun: c 4 locus lin quan n hin tng phn ly khng bnh thng l
1 locus trn vai di ca NST s 3 nm gia 2 ch th R11D12 v R25E1; 2 locus R13D5 v
R13E6 nm cch xa nhau trn NST s 6 v 1 locus trn vai di ca NST s 9 gia 2 ch th
R15G4 v R15G1.

Hnh 4. th phn ly t l kiu gen trong qun th F2 ca 80 locus

Bng 3: Danh sch 6 locus phn ly khng bnh thng
Locus/ ch th

NST

S c th F2 c kiu gen
AA

AB

BB

Phn ly
lch theo

R11D12

26

42

13

6,74

R25E1

14

61

15,32

R13D5

28

41

12

6,98

R13E6

21

49

10

8,14

R15G4

27

39

13

7,02

R15G1

30

38

13

8,70

Mt s nghin cu trn th gii cng pht hin c cc gen tng t lin quan n cch
ly di truyn khi lai la (hybrid barier); v d nh: trn NST s 3 c cc gen c cng b
nh ga-2, ga-3 (Xu et al.1997), L3b v S3b (He v Xu, 2000), S9 (Xu et al. 1997), trn NST
s 6 c cc gen ga-1, ga-4 v ga-5 (Kinoshita et al. 1997); GB (Harushima et al. 2002), S5
(Chen et al. 2008), trong gen S5 tng ng vi locus R13D5 c nhn dng v xc
nh l gen gy bt dc trong php lai Japonica v Indica (gen ln s5 tng ng l gen tng
hp rng), NST s 9 c gen Zb c cng b lin quan n cch ly di truyn giai on bo t
th (Harushima et al. 2002).
Vic xc nh c cc locus gy ra hin tng cch ly di truyn c ngha rt ln trong
phng php lai to gia cc ging la nh vic loi b cc gen lm gim t l u ht. Kt
qu thu c trong nghin cu ny l thng tin hu ch cho cc nghin cu su hn trong lp
bn gen hu ch cy la phc v cng tc chn to ging bng ch th SSR.
4. Kt lun
Da trn kt qu kho st genome, 80 ch th SSR a hnh gia 2 ging KD18 v IR24
c xc nh.
Bn vt l v bn lin kt ca 80 locus vi 12 nhm lin kt c xy dng cho
qun th F2. y l c s trin khai cc nghin cu lp bn cc gen hu ch (nng sut
cao, khng bnh v tnh thch ng rng) trong ging KD18.

Pht hin 4 locus c hin tng phn ly khng bnh thng. Kt qu ny l c s cho
nhng nghin cu tip theo sau ny.
Summary
This research utilized 93 F2 individuals derived from cross between KD18 and IR24
variety to construct linkage map for mapping agronomic QTL/gene in KD18. Result of
genome survey by SSR marker revealed 80 polymorphic markers for KD18 and IR24. Using
BLAST (Basic Local Alighment Search Tool), the physical positions of locus were located
and the physical map of 80 polymorphic locus was constructed. Based on genotypic
segregation of 80 locus in F2 population, genetic distance (centiMorgan) was caculated by
Kosambi function and linkage map was constructed with MAPMAKER3.0 software. Total
length of linkage map was 2.553,7 cM. Analysis of segregation distortion by Chi-square test
(P<0.05) identified 4 loci that may be corresponded to reproductive barriers, either
gametophytic or zygotic genes. Infromation of this study will facilitate next-step study to
map QTL/gene associated with disease resistance and abiotic tolerance in KD18 variety and
also, this is preliminary research about genetics of reproductive barriers in rice.
Keywords: F2 population, linkage map, segregation distortion, SSR marker.
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