KHOA HOC C 6 N G N G H £
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B A I V D O O T LTilXIH KHAIMG OAO OIV U LUA
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Trong nghidn cuu nay da tien hanh iSp ban dd QTL tinh khdng dao on a liia sir dung qudn th^ F6 nhan ducyc tir to hgp lai giOta gidng CR203 va T4m thorn. Kdt qua nghidn eiiu da xay dimg ducyc ban dd li^n kdt ciia 184 chi thi phan tii SSR phSn bd tren 12 nhi^m sdc the liia voi tdng chi^u dai la 1652,9 cM va khoang each tmng binh giua cae ehi thi la 9 cM, khoang each gdn nhdt va xa nhdt giira 2 chi thj tuong ling la: 0 cM va 18,2 cM.
Bdn QTL khang d^o 6n da dupe phat hi^n va iSp ban do gdm: qSLASTl-Sa-TXC, qBLASTl-Sb-TXC, qBIASn-9-TXC, qBLASTl-ll-TXC. Chi thi gdn nhdt a hai ddu mdi QTL tirorffe ung la: RM3223-RM6358;
RM2334-RM6329. MRG4657-RM434 vk RM20-MRG0761. QTL qBLASTl-11-TXC co chi sd LOD vk phdn b-am dong gop toi kidu hinh cao nhdt trong sd cac QTL dupe xac dinh, day la QTL chinh tac dpng l^n tinh khang dao 6n o liia.
Tir khoi: Tinh khang d^o dn, khang dao on dlua, l$p ban dd QTL.
I. DAT VAN DE
Nghidn cuu, lap ban dd QTL tinh khdng dao on a Ilia da dupe tien hdnh tir nam 1991. Den nay da ed khoang vdi chuc QTL dupe xdc dinh va lap ban dd trdn nhidm sde the trong he gien liia (Yu, 1991; Zhu et al., 1992; Wang et al., 1994; Zheng et al., 1995;
Chen et al., 1999; Fuji et al., 2000; Zenbayashi et al., 2002; Sallaud et al., 2003). Vide lap ban do QTL ed y nghia rat quan trpng trong eong tac chpn tao gidng Ilia khdng benh, bdi nhumg thong tin ve ban dd QTL se giiip chiing ta Idm chu vide khai thdc mpt edeh hidu qua cdc QTL khang benh phuc vu cho eong tdc chpn tao gidng khdng. Sau khi lap ban dd, ba gien khdng dao on: Pi-1, Pi-2 va Pi-3 dupe qui tu vdo gidng Ilia Co39, mpt gidng nhidm benh de tao ra ddng liia mang 3 gien khang ndi trdn. Ddng liia nay da thd hidn tinh khdng eao vdi nhieu chiing dao dn khdc nhau va dang dupe su dung ldm vat Udu khdi dau eho eong tae chpn tao gidng liia khang benh a nhidu nude trdn the gioi va b Viet Nam.
Trong sd eae gidng liia dupe kidm dinh tinh khdng benh dao dn b Vidt Nam, Tam tham cd khd nang khang rat cao vd nd thue su la ngudn gien qiiy trong tao gidng liia mdi. Tuy nhidn, lap ban dd gien khdng bdnh dao dn a gidng liia nay vdn ehua dupe tien hdnh. Chinh vi vay, trong nghidn ciru da tidn hanh phan tieh, lap ban dd QTL tinh khang dao on qua su dung quan the F6 tu to hpp lai giua gidng liia Tdm tham va gidng liia CR203 phuc vu eho vide bao tdn va khai thac hidu qua ngudn gien khdng bdnh a gidng Ilia Tam thorn.
H. VAT LI$JVA PHUONG PHAP
1. Vdt lidu nghidn cuu
Tdm tham la gidng liia miia dia phuong cd thdi gian sinh trudng khoang 160 ngdy, cay cao trung binh khodng 151 cm. Bdn canh nhiing dac tinh chat lupng tdt, gidng liia ndy cdn cd khd nang khdng dao on cao (bang 1).
CR203 Id gidng liia ed chieu cao tir 95 - 100 em, ed thdi gian sinh trudng 150 160 ngdy vdo vu xuan chinh vu, 120 - 130 ngdy vdo vu xuan mupn vd 115 120 ngdy vdo vu miia. Nang suat vdo khodng 50 - 65 ta/ha. CR203 Id gidng nhidm dao on nang khi dupe lay nhidm vdi cdc chung nam bdnh hoae trong dieu kidn cd dich bdnh phdt trien (bang 1).
2. Phuong phdp nghidn cihi a. Tao quan theF6
Hai gidng liia Tam thorn vd CR203 da dupe lai vdi nhau dd tao quan the F6 (quan the RIL). 250 ddng F6 da dupe trdng dd thu mdu tach ehiet ADN va ddnh gid phan ung bdnh (tinh khang/nhidm bdnh).
b. Danh gia tinh khang binh
Cdc cay liia bd, me va ddng F6 dupe bd tri thi nghidm dd ddnh gid phan ling bdnh vao vu dong xudn nam 2008 voi 3 lan nhdc lai. Phan ling bdnh eiia moi cay liia bd me va ddng F6 dupe ddnh gid theo thang didm ciia IRRI tir miic khdng den nhidm benh tuong ling voi thang diem tir 0 dd'n 9.
c. Phan tich ADN
La 4 tuan tudi eiia cay liia bd, me va ddng F6 da duoc thu de tach chief ADN. ADN cua Id liia duoc Trung tam Tai nguyen Di truySn thirc vat
KHOA Hpc C 6 N G W G H I
tach ehiet bang phuong phdp sir dung CTAB ciia Murray vd Thompson (1980).
Nhdn dang ADN ciia liia dupe tidn hanh theo phuong phdp PCR sir dung mdi SSR ciia McCouch vd es. (2002). PCR dupe chuan bi vdi thd tich mdi phan ling Id 20 pi pha trdn gdm: 50 ng ADN khudn; 0,5 pM ciia mdi mdi ngu(?c vd xudi; 200 pM eiia A, T, G vd C; 1,5 mM MgCl2; 0,5 don vi enzim Taq; 2 pi buffer ciia enzim Taq; sau dd didu ehinh bang nuoc cdt Idn 20 pi.
Chuang trinh chay PCR gdm ede bude: bude 1:
94''C trong 5 phiit; bude 2. 94°C trong 1 phut, 55"C trong 1 phiit; bude ndy lap lai 53 lan; budic 3. llK trong 2 phut; bude 4. 72''C trong 10 phiit.
Sdn pham PCR ducfc didn di trong gien agaroza 3% su dung dem didn di 0,5 TBE vd sau dd dupe nhupm Cling ethidium bromide vd chup anh dd ghi nhdn hinh dnh didn di.
d. Phin tich dir lieu
Da lieu ve kidu gien (nhan dang ADN) vd kidu hinh (phan ling bdnh) dupe sir dung de xdy dung ban dd Udn ket eiia ede ehi thi phan tii vd phdt hidn QTL.
Chuang trinh MAPMAKER/EXP ver. 3.0 (Lander et al., 1987) dupe sir dung de xay dung ban dd lidn ket eiia eae chi thi SSR trdn nhidm sdc the liia, khoang each giiia cdc ehi thi SSR dupe tinh bang don vi centimorgan (cM). Cac nhdm Udn kdt chi thi SSR vd vi tri cua nd trdn nhidm sde thd liia dupe xdc dinh dua vdo ban do Udn ket eiia ede ehi thi SSR b Iiia (McCouch etal, 2002).
Chuang trinh MAPMAKER/QTL ver.1.1 (Uneoln et al., 1993) dupe sir dung dd xac dinh QTL qui dinh tinh khdng dao on or liia. De xdc dinh QTL, nhiing vimg ed ehi sd LOD Oog-likelihood) Idn hon 2.0 thi b dd dupe xem nhu Id cd mpt QTL.
QTLs dupe dat tdn ea ban dua trdn dd nghi ciia McCouch et al. (1997) va Tabien et al. (2002), vi du:
tdn ciia mdt QTL la: qBIASTl-3-TXC ed nghla la QTL Udn qua ddn bdnh dao on la, ndm trdn nhidm sac thd sd 3; 9: la QTL, BLAST, la dao dn, / la la, 3 Id nhidm sde thd sd 3 va TXC la vidt tdt eiia cum tir Tam thorn XCR203"
IL KET QUA V A T H A O LUAN
1. Ddnh gid phan ung bdnh ^ Kd't qua ddnh gid phan ling bdnh trdn la liia da
cho thay, gidng Tam tham ed kha nang khdng eao.
miie dd phan ung bdnh ed didm trung binh la 1,67.
Trong khi dd gidng liia CR203, bidu hidn la mdt gidng nhidm bdnh, mire dp phan ung bdnh tmng binh ed didm Id 7,33. Ddi vdi nhirng ddng liia F6 miitc d^ bdnh tu 0,33 ddn 9 didm. Tdn sd khdng eua F6 dupe ehi ra b hinh 1. Khodng 1/2 sd ddng liia cd gid tri <3dnh gid nhd hon didm 4, cdc ddng liia cd didm nhu vdy dupe xem nhu Id khdng bdnh (R); 1/2 ddng Ilia cdn lai ed gid tri cao hon didm 4, nhirng ddng liia ndy dupe xem Id ddng nhidm bdnh (S). Ty Id giiia ddng khdng vd nhidm bdnh xdp xi 1:1, ty Id nay phii hpp vdi phdn ly ly thuydt (hinh 1). Theo kdt qua phan tieh ndy it nhat cd mpt gien khdng chinh trong gidng Ilia Tdm tham.
Bdng 1. Phdn ling bdnh ciia mOt sd gidng liia vdi cdc chiing dao 6n ed ddc tinh eao
Gidng liia Du Di Hung Tdmthom
Du eao cay Chidm bde Co39 CR203
Chung ndm 1*
S M S M S
s
2*
S R R R S S
3*
S R S R S S
4*
R R R S
s
*1:P0&6, 2: CBN9219-25. 3:IK81-3, 4: V86010. R:
khang, S: nhidm, M: khang trung binh, -: khong co dir lieu.
2. Xiy dung ban dd Udn k^t
335 ehi thi phan tu SSR phan bd trdn 12 nhidm sdc thd eiia liia da dupe su dung de phan tieh da hinh giira 2 gidng liia Tam thom va CR203 (hinh 2). 184 ehi thi da cho da hmh giiia 2 gidng Iiia ndi trdn, chid'm ty Id khoang 55%.
184 ehi thi phdn tii eho da hinh giua hai gidng Ilia da dupe sii dung dd nhdn dang ADN or F6 (hinh 3). Kidu nhdn dang ADN cua gidng Tam thom dupe ma la: A, ciia gidng CR203 ma la: B; nhung ddng F6 cd nhdn dang gidng nhan dang ADN ciia gidng Tam thom dupe ma Id A, gidng nhan dang ADN eua gidng CR203 dupe ma la: B. Dii lidu ADN eiia eay bd, me va ddng F6 dupe sii dung dd xay dung ban dd lidn kdt eiia cae ehi thi phan hi, sur dung chuang trinh MAPMAKER/EXP.
Trdn ban dd Udn kdt cae ehi thi phan tii SSR phan bd trdn 12 nhidm sde thd vdi tdng chidu ddi Id 1652,9 cM, khoang each trung binh giua ede ehi thi
KHOA HOC CdNG NGHl Id 9 cM, khoang edeh xa nhat giira hai ehi thi Id 18,2
cM vd gdn nhat Id 0 cM (hinh 4). Trat tu sdp xdp eiia ede ehi thi phdn tu SSR trdn mdi nhidm sde thd a nghidn eiiu nay eung tuong xiing vdi trat tu sdp xdp cua cdc chi thi SSR trdn ban do lidn ket a liia da dupe McCouch vd es. (2002) vd Chuang trinh genom liia
(RGP) eong bd.
T^iTifTT t f - i o f rT»
Hinh 1. Phan ling bdnh dao 6n ciia cdc gidng liia Tam tham, CR203 vd cdc ddng F6 dupe ddnh gid
theo thang didm cua IRRI
Cdc nhidm sde the: sd 1 ed sd lupng ehi thi Id 20, vdi tdng khoang edeh cdc ehi thi Id 181eM; tuong tu ede nhidm sdc the sd 2, 3, 4, 5, 6, 7, 8, 9,10,11 vd 12 cd sd ehi thi phan tii vd tdng khodng edeh tuong ling Id: 16 vd 166; 15 vd 158,1; 13 vd 123; 14 vd 124,8; 15 vd
124,4; 14 vd 121,8; 15 vd 116,8; 18 vd 131,1; 13 vd 133,4; 16 vd 127,1; 15 vd 145,4 (hinh 4).
3. Phan tich xdc dinh QTL
Dir lidu kidu gien (nhan dang ADN) vd phdn ling bdnh (hinh 4) da dupe sir dung dd phan tieh xdc dinh QTL sii dung chuang trinh MAPMAKER/QTL ver.1.1.
Kdt qud phdn tieh da xdc dinh dupe 4 viing ed ehi sd LOD Idn ban 2; nhu vdy da ed 4 QTL dupe xdc dinh qui dinh tinh khdng dao on trong qudn thd F6 tao lap giira gidng Tdm thom vd CR203. Cdc thdng sd: gid tri LOD, phdn tram tdc ddng, vi tri nhidm sde thd vd cdc chi thi SSR bidn (ehi thi ndm gdn nhdt b hai bdn QTL) eiia cdc vi tri mang gien (QTL) dupe thd hidn trong bang 2. Cdc vi tri mang gien dupe dat tdn: qBLASn-3a-TXC, qBLASTl-Sb-TKC, qBLASTl-9-
TKCvk qBLASn-11-TXC. 3 QTL, gdm qBLASTl-3a- TXC qBLASTl-3b-TXCvk qBLASTl-ll-TXC cb gid tri AE dm, gid tri ndy thd hidn gidng CR203 da ddng gdp alen ldm tang tinh khdng dao on, nhu vay mac dii Id gidng liia nhidm bdnh nhung khi cd sir tuong tdc alen vdi gidng khdng Tdm tham nd gdp phdn ldm tang tinh khdng bdnh trong qudn the. Trong sd cdc QTL da dupe phdt hidn, qBLASTl-11-TKC ed chi sd LOD Idn nhat Id 17,34 vd cd phdn tram ddng gdp tdi kieu hinh Id 37%, dieu ndy chiing td nd ddng vai trd chinh qui dinh tinh khdng dao on vd ciing phii h(7p viA ket qud phan tich phan ling bdnh eho thay ed it nhat mpt gien khdng ehinh qui dinh tinh khdng dao on b gidng Tdmthom.
QTL qBLASn-3a-TXC qBLASn-3b-TKC qBLASn-9-TKC qBLASTl-ll-TXC
Bang 2. Cdc QTL dupe p Cdc chi thi SSR bidn RM3223-RM6358 RM2334-RM6329 MRG4657-RM434 RM20-MRG0761
idt hidn Udn quan Nhidm sac thd
3 3 9 11
ddn tinh khdng dao 6n Id AEa
-0,439 -0,484 0,444 -1,297
LOD 2,622 2,514 2,776 17,340
Var. exp. (%)'•
8,8 7,7 9,1 37
" Hieu qua di truyen tang them do sir tuong tac giua 2 alen cua gidng Tam thom va CR203. AE co gia tri am cd nghia la alen lam tang kha nang khang benh den tir gidng CR203. * Ddng gop cua QTL len kha nang khang benh.
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7
Hinh 2. Nhdn dang ADN dd xdc duih su da hinh giiia hai gidng liia Tdm thom vd CR203 bang cdc cap mdi SSR. Cac cot 1 va 3 Id gidng Tdm thom; c6t 2 vd 4 Id
gidng CR203; cot 5 la ADN ladder
Hinh 3. Nhin dang ADN ciia nhiing eSy Ilia bd, me vd F6 vdi mdi SSR. Cot 1 la OX175; cot 2 la gidng liia Tdm thom, c6t 3 Id gidng liia CR203, ede cdt tu 4 ddn
17 Id cdc ddng F6
KHOA HOC C 6 N G N G H |
lU. KET LUAN VA KIEN NGH! thi Id 9 cM; khoang edeh gan nhdt vd xa nhat giira 2 Gidng Ilia Tdm tham cd khd nang khdng bdnh ^hi thi tuang ling Id: 0 cM vd 18,2 cM.
dao on tdt, gidng CR203 Id gidng liia nhidm nang vdi Bdn QTL khdng dao dn da dupe lap ban dd dao on. Cdc ddng liia F6 cd tan sd phdn bd phan ling gdm: qBLASTl-3a-TKC, qBLASTl-3b-TXC, qBLASTl- bdnh tudn theo phdn bd chuan "hinh ehudng" vd qui 9-VCC, qBLASrn-ll-TXC. Chi thi gdn nhat b hai dau ddi didm ddnh gid bdnh vd khdng/nhidm ed ty Id mdi QTL tuang ling Id: RM3223-RM6358; RM2334.
phdn ly tuang ling Id 1:1. RM6329; MRG4657-RM434 vd RM20-MRG0761.
Bdn dd lidn kdt eua 184 ehi thi phdn tii SSR QTL qBLASTl-ll-TXC co ehi sd LOD vd phan phdn bd trdn 12 nhidm sdc the liia vdi tdng chidu ddi tram ddng gdp tdi kidu hinh cao nhat trong sd cdc Id 1652,9 cM vd khoang edeh trung binh giira cdc chi QTL dupe xdc dinh, ddy Id QTL chinh tdc ddng len
tinh khdng dao on b liia.
CI
. RM6672 RM6324 . RM1167 -RM6277 -RM1220 . RM663 - RM646 - RM1287
• RM3412
• RM5385 RM5S38 RM5853 - RM591 - RM7202 . RM129 - RM126 - RM538 RM329 . RM348
" RM536 C2
. RM7033 - RM6616 , RM 3294 - RM5862 - RM5340 - RM7288 . RM1234 ' RM4499 RM8252 RM5363
• RM1920
•RM3685 - RM3421 - RM3302
• RM3B92 RM3850
C3 CA C5
RM2334 .— RM6329
RM3166 RM6970
— RM6084 -— RM3585
C6
RM4108 RM7072
•RM7576 RM6433 RM3716 RM6080 RM3223
• RM6358 RM5172
RM24ie RM595 RM568 RM6314 RM5951 RM6172 RM186 RM 1 223 RM6590 RM1 703
RM3836 RM1153 RM127
_ _ _ _ - RM3529 , RM2010 _ _ RM3345
RM3328 - RM6082
RM3437
" --RM7363
RM3663 RM3870
~~~—~ RM5970
" RM2357 RM1054
RM8106 RM1369 -RM6536 RM2126 -RM4128 RM1925 RM330 RM3187 RM4447
• RM7434 RM6071 - RM3307
RM6458 RM5463 - RM3509
C7 C8 eg C10 C11 012
RM5271 RM7545 RM2887
RM5352 RM6016 RM5494
MRe4479 RM235 MRG1197
[ ] Dp dai 10 cM H Ky hieu QTL
Hinh 4. Ban dd Udn kdt ciia cdc chi thi phdn tii SSR vd cdc QTL khdng dao 6n dupe xac dinh qua phan tich qudn thd F6 ciia td hgp lai giiia gidng Itia Tdm thom vd CR203
KHOA HOC
C 6 N G N G H ITAI UEU THAM KHAU 6. Sallaud C , M. Lorieux, E. Roumen, D.
1. Fukuoka and S. K. Okuno (2001). QTL Tharreau, R. Berruyer, P Svestasrani, O. Garsmeur, analysis and mapping of pi21, a recessive gene for ^- (^hesquiere, J.-L. Notteghem (2003). Identification field resistance to rice blast in Japanese upland rice. ^^ fiv<^ " ^ ^ blast resistance genes in the highly blast- Theor. Appl. Genet. (2001) 103:185-190. resistant rice variety IR64 using a QTL mapping
2. McCouch S. R., Cho Y. G., Yano M.. Paul E., '''''''^- '^^^"^- ^^^^- ^'^^"^^- ^^^^^ 106:794-803.
Blinstrub M., Morishima H., Kinoshita T. (1997). 7. Tabien R. E., Li Z., Paterson A. H., Report on QTL nomenclature Rice Genet Newslett Marchetti M. A, SUinsel J. W., Pinson S. R. M.
14:11-12. (2002). Mapping QTLs for field resistance to the rice 3. McCouch S. R., Teytelman L, Xu Y., D)bos ^^'''^ P''^^''^'^" ^ ^ ' evaluating their individual and K. B., Clare K., Walton M., Fu B., Maghirang R. Li ^""^^'"f^^ ""1'^^ ' " '"^P'-^^^d varieties. Theor. Appl.
7 ^ir.nr yj TU.. f^ 1.' T V A/. T- 11 . Gcuct. (2002) 105:313-324.
Z., Xing Y., Zhang Q., Kono 1., Yano M., Fjellstrom
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Nucleic Acids Res 8: 4321-4325. 9. Zenbayashi K., T. Ashizawa, T. Tani, S.
5. Nguyen T. T. T., Koizumi S., La T. N., Zenbayashi S. K., Yasuda N., Imazaki 1., Miyasaka A
(2006). Pi35 (t), a new gene conferring partial resistance to leaf blast in the rice cultivar Hokkai 188. Theor. Ai^pl. (ienet. 113 (4), pp.697-704.
Koizumi (2002). Mapi)ing of the QTL (quantitative trait locus) conferring partial resistance to leaf blast in rice cultivar Chubu 32. Theor. Appl. Genet. (2002) 104:547-552.
QTL MAPPING OF BL\ST RESISTANCE IN RICE
La Tuan Nghia Summary
The F6 lines, which generated from the cnjss of Tam thom and CR203 culUvars, were used to map QTL controlling the blast resistance in rice. The result of phenotyping showed that Tam thom is tlie resistance while CR203 is susceptible to blast fungus; the disease reactions of F6 lines followed the standard distribution and suggested there was a major gene controlling the blast resistance. 184 SSR markers showed polymorphism between the parent cultivars used to construct the linkage map based on genotyping of F6 lines. The linkage map with 1652,9 distance total was constructed, average distance between SSR markers is 9 cM, farest and nearest distances between two makers are 0 cM and 18.2.cM, respectively. Four QTL were detected to link to blast resistance. These QTL wei-e located on the chromosomes: 3, 9 and 11, and they were named: qBLASTl-3a-TXC, qBLASTl-Sb-TXC, qBLASTl-9-TXC. qBLASn-ll-TXC, and the flanking markers of 4 QTL are: RM3223-RM6358, RM2334-RM6329, MRG4657-RM434 and RM20-MRG0761 respectively. QTL qBLASTl-ll-TXC had largest LOD and the percentage contributed to the phenotypic variation, this suggest it is major QTL managing the blast resistance in rice.
Keywords: Rice blast, blast resistance in rice, QTL mapping.
Ngudi phan bidn: TS. Nguydn Thi Thanh Thuy
