KHOA HOC CdNG N G H £
X A Y DIJIXIG B d CHI T H | PHAIVI T U DAC H I E U DE DAIMH G I A , PHAIM DIET CAC GldlMG L U A H d T R O CHG
K H A O IMGHIEIVI D U 5
Luu Minh CiicS Luu Thj Nggc Huydn', Nguydn Diic Thinh^ VQ Due Quang' T6MTAT
Xac dinh diing cac ddng/gidng cSy trong nong nghiep 1^ mpt cong vi^c quan trpng trong chc?n tao gidng cay frong va co tinh th6i su frong vi^c bao hp quyen tac gia. Dd cong nh^n gidng nich cin tidn hanh khao nghiem DUS (bao gdm khao nghidm tinh khac bi^t - distinctness, tinh dong nhit - uniformity va tinh on dinh - stability). Tren thuc te co nhidu gidng cua cac tac gia khac nhau nhung co hinh thai rat giong nhau duoc dua vao khao nghidm, gSy ra su franh cai ve ban quyen gidng ma chua co each phan dinh r6 rang. Dd giai quyet van dd thuc te dat ra trong qu^ trinh khao nghidm DUS Udn tcM dang ky bao ho ban quyen giong, vif c xSy dung m6t bp chi thi phan tu dSc hieu dd danh gi&, phSn biet gidng lua 1^ rat thiet thuc va cap bach.
Tong sd 412 chi thi dup«c sir dung, trong do gdm 256 chi thj SSR nim rai rac tren 12 nhidm sdc thd, nhieu gien quan frpng nhu QTL chju man, chiu ngSp, khang riy nSu, bac la, khang dpc nhom, chiu phen, chiu ban... da dug«c iSp ban dd su dung cac chi thi nay; 156 chi thj InDelduqc thiet kd dua tren su so sanh trinh tu he gien cua giong liia Indica 93-11 va gidng lua Japonica Nipponbare; co the xac djnh dupe su da hinh tir cac dot bien them/bcrt Onsertion/delection) frong h$ gien liia. Khao sat 412 chi thj SSR tr^n tong sd 260 giong Ilia da lap ra bp gom 30 chi thj dac bifu, c6 the diing cho vi^c danh gia phan bift cac giong liia h6 trp cho khao nghiem DUS tai Viet Nam.
Tirkh6a: Ban quyen gidng, chl thi phin tu, DUS (Distinctness, Uniformity, Stability), khao nghiem, lua.
LDilTVANK
Nam 1961, Hiep hgi qudc te Bio hd Gidng cay frdng mdi ( UPOV) dugc tiiinh lap. Nam 2007 Viet Nam chinh tinic fro thinh thinh vidn thii 63. Do dd vide ip dung bio hd quyen tic gii gidng ciy frdng mdi frdr tiiinh yeu ciu bit budc khi nude ta tham gia Td chiic Thuong mai Qudc te (WTO). Xic dinh diing cic ddng/ gidng ciy trdng ndng nghidp l i mdt cdng vide quan trgng trong chgn tao gidng ciy trdng vi cd tinh thdi su trong vide bao hd quyen tic gii. De cdng nhin gidng mdi cin tien hinh khio nghidm DUS (bao gdm khio nghidm tinh khic bidt - distinctness, tinh ddng nhit - unfformity v i tinh dn dinh - StabiUty). Gidng mcii mudn dugc bio hdphii thoi man 5 tidu chuan: tinh mdi vd thuong mai, tdn goi phu hgp vi phai khic bidt, ddng nhat v i dn dinh.
Trong dd tinh khic bidt, tinh ddng nhat v i tinh dn dinh li cic tidu chuan ky thuit, dugc xic dinh tiidng qua khao nghidm DUS, trong dd gidng mdi dugc gieo frdng cimg v i so sinh vdi cic gidng hidn cd nham dinh gii su khic bidt giiia chiing. Trung tim Khio kiem nghidm Gidng, Sin pham Ciy frdng vi
'Vifn Di tmyen Nong nghifp, Vifn Khoa hpc Nong nghi?p Vi?t Nam
^Vi^n Cong nghf Sinh hpc, Vi?n Khoa hpc va Cong ngh?
Vi?t Nam
Phan bdn Qudc gia (TTKKNG) thudc Cue Trdng frgt, Bp Ndng nghiep vi Phit trien Nong thon, li don vi diu mdi ve khio nghidm, kiem nghiem gidng, sin pham cay frdng vi phin bdn. Trong 10 nam gin day, mdi nam TTKKNG tien hinh khio nghidm khoing 100 gidng lua. Khi khao nghidm, thudng gap phii tinh trang (5-10 trudng hgp mdi nam) cic gidng dua ra khio nghiem cd cic tinh trang hinh thii (doi khi c i tinh frang hda smh) gidng nhau.
Ciu hdi dat ra li thuc chat chiing thudc ciing mdt gidng hay chiing thudc cic gidng khic nhau?
Cic tidu chi khio nghidm DUS truyen thdng dua frdn CO so 62 tinh frang hinh thii v i hda sinh td ra chua du dd phan bidt cic gidng. Trdn tiiuc te cd nhidu gidng cua cic tic gia khic nhau nhung cd hinh thii rit gidng nhau dugc dua vio khio nghidm, giy ra su tranh cai ve bin quyen gidng m i chua cd cich phin dinh ro ring. Chi cd xic dinh bing phuong phip sinh hgc phin tir frdn co so dac trung ADN mdi cd tiie giii quydt dugc van de niy. Dac trung ADN li chi tidu quan trgng v i cin thiet vi nd giiip dinh gii chinh xic cho vide giim dinh vi phin bidt mdt gidng cay frdng mdi. Cic phuong phip ADN diing cho khio nghidm DUS ciing da v i dang bit diu dugc ip dung tai mdt sd qudc gia. Kdt qua cua vin tay ADN (DNA fingerpruiting) nhin dugc tiidng qua phin tich PCR ciing vdi thdng tin ve p h i he cdng vdi cic tinh
KHOA HOC C 6 N G NGHfi
trang da gien quan trgng cd y nghia ndng hgc dugc thira nhan nhu la co so- khoa hgc vi tiiuc tidn khich quan nhat di md ta vi dang ky gidng. Dd giai quydt van di thuc td dat ra trong qua trinh khao nghiein DUS tien tcii dang ky bao hd ban quydn gidng, viec xay dung mdt bg chi thi phan tii dac hidu dv dinh gii, phan biet gidng lua la rat thidt thuc va cip bich.
U. VAT UEU VA PHUONG PHAP NGHIEN CUU
1. V^t Udu
Tap doan 260 gidng lua ducrc tiiu tiiap tir nhidu ngudn, bao gdm: 70 gidng lua ndp, 34 gidng liia nuong, 34 gidng lua thom, cic gidng lua td khac tir nhieu ngudn khic nhau, gidng dja phuong, gidng nhip noi, gidng mdi.
412 chi thi SSR thich hgp cho vivc sing lgc cic chi tiij da hinh cao de lap ra bg chi thj tiiam chidu.
Cic vat tu boi chit sinh hoc phan tii chuydn dung.
2. Phuong phip
Tich chidt ADN tdng sd theo phuong phip CTAB cai tidn.
- Phuong phip PCR vcM cic mdi SSR.
Phuong phip phin tich bang ADN bing didn di frdn gel polyacrylamit 4,5% bidn tinh vi nhugm bac.
Phuong phip phin tich bang ADN bing didn di frdn gel polyacrylamit 6% vi nhudm sybersafe.
- Dinh gii DUS 40 gidng lua de kdt hgp vcii phan tich bang chi thi phin tir. Thi nghiem dugc tidn hinh theo quy pham khio nghidm tinh khic bidt, tinh ddng nhit vi tinh dn tinh ciia gidng liia lOTCN 554 - 2002.
lU. K ^ QUA VA T H A O LU/iN
1. Khio sit xic djnh sd alen vi kich thudc alen cua cic chi th; c6 tidm nang cho da hinh cao
Tdng sd 412 chi thj dugc sir dung, frong dd 256 chi thj SSR nim rai ric frdn 12 nhidm sic the, 156 chi till InDcl
Tac- gi.i Bonow vi ctv (2009), khi khio sit phan phia trc'ii (upstream) vimg bio tdn cua cic ydu to phidn ma cung nhu khi khio sit cic EST b liia, da phat hidn dugc 50 chi thi SSR mdi. Cic tic gii nay da sir dung chiing dd danh gii da dang di truydn 37 gicing lua cd ngudn gdc japonica vi indica vi da tim ra 19 chi thi SSR cd nhidu alen, cd khi nang phan bidt dugc cic gidng cd quan hd rat gin. Cic tic gia niy da di dc-n ket luan ring cic chi thi SSR cd the ddng vai trd nhu mot ngudn cung cip thdng tin b^n canh cic thong tin khic vd sir khic biet hinh thii ma Lidn bidp Bio vd Gidng ciy frdng mdi Qudc t^
(International Union for the Protection of New Varieties of Plants) da dua ra ddi vcii khio nghidm DUS (UPOV, 1991). Dua frdn cic kdt qui niy, ban dau, nghidn ciru tidn hinh khio sat 260 gidng liia thu thip tir nhidu ngudn khic nhau ddi vcii 81 chi thi SSR rai ric frdn 12 NST, cd sd lucmg tir 3-8 alen; 6 chi thi nim frong vimg dieu khien cua mgt sd gien lua chon ddi vdi nhirng gien cd chira mdi vd tinh
(microsateUite) gdm hai don vi lap lai tir 10 lin fro len thong qua M-WS box genes; 13 chi tiij tir dir Ueu EST frdn ngin hing gien (Gene Bank) tir NCBl thong qua vide tim cic trinh tu EST cd chua cac microsatellite gdm 3 nucleotit dugc lap lai it nhat 6 lin fro Idn.
Stt 1 2 3 4 5 6 7 8 9 10 11 12 13
Locut RMI RM259 RM495 RM283 RM312 RM237 RM431 RM5 RM718 RM485 RM154 RM174 RM452
Nst
2 2 2 2
Bing 1. Danh sich 100 chi thj SSR dinpc chgn lc?c ban Stt
26 27 28 29 30 31 32 33 34 35 36 37 38
Locut RM307 RM255 RM551 RM241 RM124 RM5757 RM6005 RM6997 RM122 RM507 RM413 RM159 RM169
Nst 4 4 4 4 4 4 4 4 5 5 5 5 5
Stt 51 52 53 54 55 56 57 58 59 60 61 62 63
Locut RM70 RM455 RM18 RM408 RM25 RM547 RM223 RM284 RM433 RM447 RM316 RM105 RM257
Nst 7 7 7 8 8 8 8 8 8 8 9 9 9
Stt 76 77 78 79 80 81 82 83 84 85 86 87 88
diu
Locut RM144 RM20A RM19 RM277 RM17 RM4A MADSl MADS3 MADS4 MADS8 MADSll MADS13 EST17Nst 11 12 12 12 12 12
KHOA HOC CdNG NGHl
14 15 16 17 18 19 20 21 22 23 24 25
RM300 RM262 RM341 RM263 RM6 RM250 RM266 RM22 RM338 RM16 RM514 RM148
2 2 2 2 2 2 2 3 3 3 3 3
39 40 41 42 43 44 45 46 47 48 49 50
RM164 RM161 RM31 RM39 RM178 RM133 RM510 RM585 RM276 RM162 RM481 RMll
5 5 5 5 5 6 6 6 6 6 7 7
64 65 66 67 68 69 70 71 72 73 74 75
RM215 RM333 RM474 RM222 RM271 RM171 RM484 RM21 RM167 RM552 RM536 RM287
9 10 10 10 10 10 10 11 11 11 11 11
89 90 91 92 93 94 95 96 97 98 99 100
EST19 EST20 EST22 EST24 EST27 EST31 EST33 ESTr36 EST44 EST45 EST46 E.^50 Cic chi thi frong hop gien MADS cd mot sd
chiic nang quan frgng frong su phit trien cua thuc vat nhu dieu khien qui trinh bien ddi tir giai doan sinh duong sang giai doan sinh thuc, xic dinh ciu tao vi td chiic hoa, dieu khidn qui frinh chin ciia qui, hat, lidn quan den thdi gian sinh trudng cua ciy...
^ •» w
}0
Wfm,*.^r.^ifw^%
l iHinh 2. Phan tich alen cua 1^333 frc^n gel polyacrylamit, vcri marker 25 bp
~ - . m^jm-tji
» "^
- . «
1 C t_<(^ • T T * * J -
4 (TV » , * ^
* • - « . « • «^«i ^^•»-»-7- «-#« 4i > Jf *, -f-*- •?Mfc«"-»
Hinh 1. Phin tich alen cua RM17 fr-dn gel polyacrylamit Cic miu dugc nap vio gidng thinh 3
lugt vdi marker 25 bp tuong ung li Ml, M2, M3 MADSl Udn quan den su dieu khidn boat ddng sdm cua mim phdi tbii hai vi thii ba. MADS2 vi 4 lidn quan den gien quy dinh kich thucic, hmh ding vi su khic nhau cua cic td chiic trong phdi thii cip. MADS 3 vi 58 Udn quan den chiic nang vugt trdi cua cic loai mim phdi khic nhau. MADS 5, 7 vi 8 Uen quan den tiidi gian nor hoa. MADS 11 chi the hidn vai frd frong su ra hoa khdng xic dinh. MADS 13 didu khidn su phit frien cua phdi vi hat. MADS 18 tiiiic day su phit trien sdm ciia phdi.
Hinh 3. Phan tich alen cua RM481 fren gel polyacrylamit vdi ladder (thang) 25 bp
Qua budc sing lgc vcii 100 chi thi, ti Id vd tin suit cic alen dugc tdng kdt lai b bing 2.MADS 1 Uen quan den su didu khien boat dgng sdm cua mim phoi thu hai vi thii ba. MADS 2 vi 4 Udn quan den gien quy dinh kich thucic, hmh ding vi su khic nhau ciia cic td chirc frong phdi thir cip. MADS 3 vi 58 lien quan den chirc nang vucpt trdi ciia cic loai mim phdi khic nhau.
MADS 5,7 vi 8 Udn quan den thdi gian nor hoa. MADS 11 chi the hidn vai frd trong su ra hoa khdng xic dinh.
MADS 13 dieu khien su phit trien ciia phdi vi hat.
MADS 18 thiic day su phit trien sdm cua phdi.
Bang 2. Tin suit cic alen ddi vdi 100 chi tiii khio sit Nhdm
Nhdm 1:
1-3 alen
Tdn chi tiii
Ml, E17, E19, E22, E24, E27, E31, E33, E36, E44, E45, E46, E50, RMI, RM4, RM16, RM18, RM39, RM70, RM124, RM133, RM144, RM148, RM154, RM159, RM161, RM164, RM167, RM169, RM171, RM174, RM178, RM215, RM222, RM237, RM241, RM255, RM257, RM262. RM263, RM271, RM277. RM284. RM300, RM316, RM338, RM341, RM413. RM431, RM433, RM452, RM455, RM474, R484, RM485, RM495,
KHOA HOC CdNG N G H |
Nhdm 2:
4 - 6 alen
RM507. RM510. RM514, RM536, RM547, RM718, RM5757, RM6005, RM6997 RM5, RM6, RMll. RM19, RM20, RM21, RM22, RM25, RM31, RM105, RM122, RM162, RM250, RM259, RM266, RM276, RM283, RM287, RM307, RM312, RM408, RM447, RM551, RM585, E20, M3, M8, Mil
Bing 3. Danh sich 49 c Stt
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Tdn mdi R1M7 R1M20 R1M30 R1M37 R1M47 R2M10 R2M24 R2M26 R2M37 R2M50 R3M10 R3M23 R3M30 R3M37 R3M53 R4M13 R4M17
li thi ind NST
2 2 2 2 2 3 3 3 3 3 4 4
el cua Shen v i ctv, 200^
Stt 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Tdn mdi R4M30 R4M43 R4M50 R6M44 R5M13 R5M20 R5M30 R5M43 R6M14 R6M30 R7M7 R7M30 R7M37 R8M10 R8M23 R8M33
da khio
NST 4 4 4 6 5 5 5 5 6 6 7 7 7 8 8 8
sit dd xi Stt 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
ic djnh chl thj cr Tdn mdi
R8M46 R9M10 R9M20 R9M30 R9M42 RIOMIO R10M17 R10M30 R10M40 R11M17 R11M23 R11M40 R12M10 R12M27 R12M33 R12M43
10 da hu NST
8 9 9 9 9 10 10 10 10 11 11 11 12 12 12 12
B ing 4. Danh sich 107 chi thi S\
Stt 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Tdn mdi SO 1160 SO 1038 SO 1091 S01132A
SO 1140 SOI160 S02013 SO2026 SO2054 SO2057B SO2081B SO3065 SO3072 SO3068 SO3010B
SO3020 SO3048 SO3065 SO3068 SO3072 SO3076B
SO3087 SO3099
NST
2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3
"S cuaC Stt
37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
lin's v i ctv, 2(
Tdn mdi SO6005A
SO6061 SO6065A SO6065B S06117 S06122 S06124 SO7053 SO7011 SO7024 SO7048 SO7053 SO7101 SO8055 SO8012 SO8035 SO8049 SO8052B SO8052C SO8055 SO8059 SO8090 SO8106
)04dak]
NST 6 6 6 6 6 6 6 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8
l i o sit d Stt 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95
i x i c dinh chi t Tdn mdi
S10016 S10026C
SI0053 S10(\58A S1(X)58B S10072 SI1033 S1132A S11117C S11004A SI1033 S11045A
SI1049 S11055A S11055B S11115 S11117A S11117C S12055 S12011B
S12029 S12030 S12041B
hi cho da NST
10 10 10 10 10 10 11 11 11 11 11 11 11 11 11 11 11 11 12 12 12 12 12
linh cao
KHOA HOC C 6 N G N G H I
25 26 27 28 29 30 31 32 33 34 35 36
SO3120 S03136 S03145 SO4058 SO4060 SO4065 SO4077B SO4097B S04128 S4129B SO5009 SO5009
3 3 3 4 4 4 4 4 4 4 5 5
61 62 63 64 65 66 67 68 69 70 71 72
S08121A SO9000A SO9003 SO9004 SO9006 SO9026B SO9040B S09()73 SO909;5A
SHXXM S10013A
S10015
8 9 9 9 9 9 9 9 9 10 10 10
97 98 99 100 101 102 103 104 105 lOG 107
S12055 S12062 S12066 S12075 S12088 S12091 S12097A
SI2099 S12109 S12019A S12109B
12 12 12 12 12 12 12 12 12 12 12
Ngoii ra, trdn 12 NST, dgc theo ban do he gien ""^^ ^ren 12 NST, nhieu gien quan trong nhu QTL (genome) cua lua, da chgn dugc tiidm hon 150 chi ^^'^ "i^"- ^^iu ngap, khing ray nau, bac la, khang tiij de sing loc chi tiii da binh cao cho bg chi tiij ^^"^ "^'om, chju phen, chiu ban... da dugc lap ban do tiiani chieu. Cac chi tiii nay boat dgng tdt va nim rii ^^^ ^^"^ ^''^^ ^^^' ^^ "'^y-
Bing 5. Danh sich 156 chi thi SSR da khio sit di xic dinh chi thi cho da hinh cao Stt
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Tdn mdi RM10055 RM10115 RM10260 RM10649 RM10655 RM10694 RM10696b RM10701 RM10711 RM10711a RM10713 RM10792 RM10793 RM10796 RM10852 RM10864 RM 10890 RM10927 RM11125 RM11438 RM11570 RM1208 RM212 RM3412b RM490 RM493 RM7075
NST Stt 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79
Tdn mdi RM5639 RM6329 RM7097 RM 16589 RM16612 RM16686 RM16842 RM335 RM3843 RM567 RM5688 RM5749 RM6487 RM6659 RM163 RM 17954 RM18161 RM18877 RM 19034 RM19122 RM249 RM334 RM3838 RM19545 RM19840 RM 19896 RM20192
NSl^
3 3 3 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 6 6 6 6
Stt 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131
Tdn mdi RM22786 RM22825 RM22897 RM23061 RM44 RM80 RM219 RM23654 RM23662 RM23759 RM23870 RM23877 RM23970 RM24013 RM242 RM24804 RM296 RM7175 RMI 126 RM228 RM24865 RM25022 RM25181.
RM25271 RM5806 RM6142 RM6691
NST 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 9 10 10 10 10 10 10 10 10 10
KHOA HOC CbNG N G H l
28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52
RM8094 RM3252 RM109 RMllO RMI 2769 RM13197 RM13(i28 RM2()8 RM211 RM3828 RM5356 RM5404 RM6318 RM 14545 RM16 RM 16236 RM186 RM227 RM231 RM3202 RM3297 RM3864 RM3867 RM55 RM5626
1 1 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3
80 81 82 83 84
<S5
<s(;
87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104
RM 20224 RM204 RM3 RM3628 RM454 RM527 RM528 RM5753 RM6654 RM7355 RMIO RM118 RMI 25 RM2 RM20848 RM21478 RM21539 RM21584 RM21645 RM248 RM3753 RM5436 RM7338 RM149 RM152
6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 8 8
132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156
RM206 RM209 RM224 RM25972 RM26063 RM26237 RM26501 RM26652 RM 27085 RM3137 RM247 RM27481 RM27593 RM27695 RM27877 RM 27933 K.\l279(i5 RM28100 RM28102 RM2.S2(kS RM28466 RM28485 RM28502 RM28746 RM7102
11 11 11 11 11 11 11 11 11 11 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 Trong sd 312 chi thi cic loai lira chgn ra ci budc
thir hai, chi nhtmg chi thi cho bang ADN ro ring vi nhidu alen da hinh mcii dugc chon cho bucic sang lgc tidp theo.
2. Thidt lap b/^ chi thi tham chi^u
tim
Cic chi thi cho bang .\1)N rci net se dugc khao sit da hinh vcii 16 - 24 - 30 - 34 - 48 gidng de chon loc cic chi tiij cho da hinh cao, tim kich thudc alen va ghi nhin bang da hinh, lira chgn de lap bg chi thi tham chieu.
Hmh 4. Khao sat cac alen cua chi tin R1M17-R5M210-S()3(X35 vcri 16 gicing (frai) va R3M53 va R4M30vdi24 gidng (j)hai) frc^n gel ix)lyacryl;unit 6% vcri ladder thimg 25 bp
Hmh 5. Khao sit cic alen ciia chi tiii RM243 (frai) va RM3468 (phai) ^d^Sp gidng frdn gel polyaciylatcii^
vdi ladder thang 25 bp
11
KHOA HOC C 6 N G N G H |
Thdng qua ket qui khio sit, da chgn dugc mgt sd cich nhau dii xa de cd the nhan biet vi ghi nhan ket chi tiii cd thd dimg de lip bd chi tiii tham chieu, cic ^"^ trdn gel polyacrylamit 4,5% nhugm bac va gel chi till niy cho bang ADN ro, kich thucic cic bang Polyacrylamit 6-8% nhuom sybersafe.
iif BIS
( 1 tt iJR
JP
Wm
mm IJ^^pl
Hinh 6. Dinh gii cic alen cua chi tiii RM21(ti-ii), RM163 (phii) trong bd chi tiii tiiam chidu vdi ladder thang 25 bp
Hinh 7. Dinh gii cic alen ciia chi tiii RM3412 fr-ong Ket hgp giiia phan tich cic chi tieu DUS kdt hgp vdi cic ket qui khio sit ci fren, da chgn ducx: bg chi thi dac hieu bao gdm:
a) Bd chi tiii dinh gii so bg (5 chi tiij): RMll, RM21, RM163. RM481, RM3412B.
b) Bg chi tiii chuan (20 chi tiii): Gdm 5 chi tiij fren: RMll, RM21, RM163, RM481, RM3412B vi 15 chi till khic: RMI, RM5, RM6, RM17, RM19, RM206, RM333, RM3252, RM3843, RM7097, RM11125, R4M13, SO1160, S11033, MADS13.
c) Bd chi till mo rong (10 chi tiij): RM210, RM223, RM341, RM7558, RM3468, RM 10827, RM19545, EST20, MADS3, MADS8.
Cho den nay, frdn the gidi da cd k h i nhieu nude sir dung cdng cu dac tnmg ADN trong khio nghidm DUS vi dang ky bin quyen gidng mdi. Tai Trung tam Qudc td Nghidn ciiu Cii tien gidng Ngo vi Lua mi (CIMMYI), ngudi ta phan bidt cic gidng ngo nhdr dac tiimg ADN (DNA profiles). Khoang 40-50 chi tiij SSR (cic chi thi cd nhieu alen) dugc sir dung nhu bd chi till tiiam chidu dugc dimg trong nghidn ciiu da dang di tiiiydn, phin bidt cic gidng ngd khic nhau, ddng tiidi phit hidn cic alen mdi trong nhirng gidng ngo dia phuong frdn toin ciu Gang v i ctv, 1999).
Hidn nay or ngd, ngudi ta da sir dung hon 20 chi thi tham chieu rii ric trdn 10 cap nhidm sic thd vdi cic kidu lap lai tii 2-5 nucleotit v i cd tii 2-5 alen (vi du:
phiWl frdn NST sd 7 cd 3 alen: 198, 202, 206 bp, hay phi070tren NST sd 5 voi 4 alen: 200, 205, 215 vi 220 bp). Nhdr cd bd chi thi tiiam chieu, ngudi ta dd ding nhin ra cic gidng ngd khic nhau dac thii o miic do phan tii ADN. Cic chi tiii SSR ti-dn ciy liia mi, liia mach, huong duong... (Roder v i ctv, 1998; Dorocicz
bd chl thi tham chidu vdi ladder tihiang 25 bp
vi Kasha, 2000; Macaulay vi ctv. 2001; Tang vi ctv, 2002) cung dugc phit frien vi sir dung cho cic nghien cim da dang di truyen, lap bin dd phan tii...
Tai Vien Di truyen chgn gidng tiiuoc Trung tam Qudc gia vd Nghien cim Hat gidng Ngu cdc vi Gidng cay frdng thuoc Vien hin lam Khoa hoc Nong nghiep Ucraina vi Vien Giim djnh Gidng cay frdng thudc Cue Bio hd Ban quydn Gidng ciy frdng Ucraina, ngudi ta da nghien ciai thinh cdng vi dua ra nhirng bg chi tiij phan tii SSR de phan biet vi xic dinh diing kieu gien ddi vdi cay lua mi, lua mach, ngo vi hudng duong. Bd chi thj niy giup xic dinh dung kieu gien cua gidng, md t i cic dac tinh di truyen cua chung vi phan inh dac diem ridng cua chiing dudi dang cong thiic vd tiiinh phin alen ciia cic locus SSR, kiem fra do sach di truyen cua gidng vi cic ddng tu phdi, dinh gii gidng lai, tiiiet lip ngan bing gidng dang ky o Ucraina vi tiiidt lap su kiem soit qui frinh chon gidng cung nhu su tiiay ddi tiiinh phin gidng frong canh tic (Cueojian vi ctv, 2004).
Nam 2005 dinh dau budc ngoat frong vide dat vin de sir dung chi thi phin tii hd frg cho khio nghidm DUS. Kwon vi ctv (2005) da sii dung mdt bo chi tiij SSR giup cho khao nghidm DUS o ciy dt. Tic gii Deniken (2005) frong cugc hgi tiiio ve "Ky tiiuit phin tii frong bio vd gidng cay frdng" or Canada nam 2005 da di xuit su dung chi tiii phan tii frong khio nghidm DUS. Sau dd, Jerko v i ctv (2008) da sir dung 28 locus SSR de phin biet 41 gidng ngd lai hd frg cho khao nghidm DUS. Tidp tiieo, mdt nhdm tic gii cua Uy ban Gidng tiiudc Hiep hdi Qudc td vd Kiem nghidm hat gidng da tien hinh thii nghidm tinh diing gidng frdn co sdr phin tich ADN, frong dd hg su dung 13 chi thi SSR ddi vdi 8 gidng diu tuong, 8 chi tiii
KHOA HOC C 6 N G N G H £
SSR ddi vcri 8 gidng lua mi, 12 chi thi SSR ddi vcri 8 gidng ngd va 8 chi thj SSR ddi vcri 10 gidng lua
(Carasini va Vicario, 2008). Nhdm tac gia nay da kdt thiic bucic thir nghidm dau tic'n va du dinh si> tien hanh buck- thir nghidm tid]! theo trircK' klii dua ra du thao vd sir dung phuong phap ADN de xac djnh tinh dimg gidng. Ngoai ra, Sayyed va ctv (2009) da sir dung 15 chi tiii SSR va 29 chi thj RAPD dv phan biet
16 kieu gien khac nhau cua hia lai. Danh gia da dang di tmyen 18 gidng lua ciia Veiu^zuela, cac lac gia Tliauna va ctv (2008) cung da sir dung 48 chi thj SSR de phan tich \'a da thu dircic 36 locus cho da hinh vcri tongsd 112 alen.
Vice sir dung bg chi thj dac hidu cho vi(v dinh gii phan biet cic gidng hia bci trg cho khao nghiem DL^S la rat c-an thicM ddi vcri ccing tic bao hg bin quyen, cong nhan gidng mdi. Qui frinh sir clung bg chi till dac hieu frong cic bucic khao nghidm DUS dang ducx- xay dung vi hoin thidn di dua vao sir dung.
IV. KET im
Da thinh cdng frong vide khio sit 412 chi thi SSR chon ra tir toin bd genom (he gien) lua de lap ra bg chi thi dac hidu cho viec dinh gii phin bidt cic gidng lua hd frg cho khio nghidm DUS bao gdm:
Bd chi thj dinh gii so bd (5 chi thj): RMll, RM21, RM163, RM481, RM3412B.
Bg chi thj chuan (20 chi thi): Gdm 5 chi thi frdn: RMll, RM21, RM163, RM481, RM3412B vi 15 chi thi khic: RMI, RM5, RM6, RM17, RM19, RM206, RM333, RM3252, RM3843, RM7097, RM11125, R4M13, SO1160, SI 1033, MADS13.
Bd chi till mdr rdng (10 chi tiij): RM210,
R M 2 2 3 , R M 3 4 1 , R M 7 5 5 8 , R M 3 4 6 8 , R M 1 0 8 2 7 , R M 19545, E S T 2 0 , M A D S 3 , M A D S 8 .
TAI UEU THAM KHAO
1. Bonow S., Von Pinho E. V. R., Vieira M. G.
C , Vosman B., 2009. MicrosateUite markers in and around rice genes: applications m variety identification and DUS testing. Crop Sci., vol. 49, pp.880-886.
2. Carasmi E., Vicario A. L, 2008. First comparative test on DNA-based methods: final report of the Variety Commitee Workmg Group. Seed
Testing International Nr. 135, pp. 26-28.
3. CiiBOjian KD. M., Bo.nKOAaB B. B., BajibBHHCKaji M. C , Ko>KyxoBa H. E., ConoACHKO A. C , 2004.
HilCHTH(l)HKaUHJl II pcrHCTpaUHfl rCHOTHnOB MaPKOii
nuiciiHUbi (Triti'eum aestivumL.), jiMMeHJi (Hordeum vulguiv L), KyKypyibi (Za maysL.), noiiconHewnmca (Hckmtiius unnuus L.) c noMouibio anajiHsa MHKpocaicjiHTiibix MapKcpoB. MemoduHecKue pcKo.MciiOaiiuu - PcaicHHfl YneHbix CoseroB KD/KHoro 6nc)ie,\iic)jioi u'-iccKoro ueiiTpa B pacreHHeBOiicTBe YAAII II MOHY; CcneKUiioHHo-reHeTHHecKoro HHCTHTyia HanHoiiajibiioro ncHxpa ceMeHosoAwecTBa H copTOH3yHennfl YAAH; HHCTHTyra 3KcnepTH3bi coproB pacTeimii rocc.ny>K6bi no oxpane npaB Ha copra pacTeiiiiii MMHarpononiiTHKH YKpaiiHbi. 17 trang.
4. Deneken G., 2(X)5. Molecular markers and DUS testing, UPOV current situation. Report of Proc.
of Seminar on the Use of Molecular Techniques lor Plant Variety Protection. Ottawa, ON, Canada, 16-17 June 2005. Canadian Food Inspection Agency, Ottawa, ON, Canada.
5. Dorocicz I., Kasha K., 2000. An analysis of polymorphism of large-motif barley microsateUites.
Theor Appl Genet, vol. 100, pp.601-609.
6. Jang C , Edmeades G. 0 . Armestead I., Lafitte H. K., Hayward M. D., Hoisington D., 1999.
Genetic analysis of adaptation differences between highland and lowland maize usmg molecular markers. Theor Appl Genet, vol. 99, pp.1106-1119.
7. Jerko Gunjaca, Ivica B., Mirko J., Hrvoe S., Antiin v., Zdravko K., Antim J., Ivan P., 2008.
Discrimmating maize inbred lines using molecular and DUS data. Euphytica vol. 161, No.l-2/May 2008, pp. 165-172.
8. Kwon, Y. S., J. M. Lee, G. B. Yi, S. I. Yi, K M.
Kim, E. H. Sob, K. M. Bae. E. K. Park, I. H. Song, and B. D. Kim, 2005. Use of SSR markers to complement tests of distmctiveness, uniformity, and stability (DUS) of pepper (Capsicum annuum L) varieties. Moi. Cells 19:428-435.
9. Macaulay M., Ramsay L , PoweU W., Waugh R., 2001. A representative, highly informative
genotyping sef of barley SSRs. Theor. Appl Genet, vol. 102, pp.801-809.
10. Roder M., Korzun V., Wendehake K., Plaschke J., Tixier M.-H., Leroy P., Ganal M., 1998. A microsateUite map of wheat Geitetics, vol. 149, pp.
2007-2023.
KHOA HOC C 6 N G N G H £
11. Sayyed H. H., Sayyed A. M. M. M., Ghorban Assessment of genetic diversity in Venezuelan rice A. N., Ahmad A., 2009. Identification of rice hybrids cultivarl usmg simple sequence repeats markers, using microsatellite and RAPD markers. Atiiean Eleetionic Journal of Biotechnolog, vol.11. No.5.
Journal of Biotechnology vo\, 8 (10) pp. 2094-2101. ^jtjl CAM ON
12. Tang S., Yu J.-K., Slabaugh M., Shiniani D., ^bdm tic gia xin chin tiianh cim on Chuong Knapp S., 2002. Simple sequence repeat map of the ^jj^^ Cong nghd Sinh hgc Ndng nghidp, Vu Khoa sunflower genome. Theor Appl Genet, vol. 105, pp. ^^^ ^dng ngh^, Bg Nong nghidp vi Phit tridn nong 1124-1136. tj^5jj ^g cap kinh phi dd thuc hidn dd tii.
13. Thaura G. H., Duna P. D., Iris P. A., Gelis T.
N., Alejandro J. P., Cesar P. M., Joe M. T , 2008.
DEVELOPING A SPECIFIC SET OF SSR MARKERS ASSISTED FOR EVALUATE THE DISTINCTNESS, UNIFORMITY AND STABILITY ON RICE
Luu Minh Cue, Luu Thi Ngoc Huyen, Nguyen Due Thanh, Vu Due Quang, Summary
Identification of the proprietary variety in agriculture is an great important work in plant breeding and in authorize protection. To recognize the new variety, it was required to test the distinctness, the uniformity, and the stability (DUS testing). Many varieties of the different authors but having similarity in almost agronomic traits. When these varieties were brought to DUS testing, it was very difficult to jugde in an exacfly words. To deal with this fact problem, development of the specific molercular markers assited for evaluate the distinctness, the uniformity, and the stability was became very urgent. Total of 412 markers were used, which included 256 SSR markers contributed along the 12 chromosomes and 156 InDel markers. The 256 markers were selected from map of several genes like brown planthopper, bacterial blight resistance, salinity, submergence, alluminium, zinc, drought tolerances... The 156 InDel markers were designed from the different sequencing information comparision of insertion/deletion mutants between the genom of Indica 93-11 and Japonica Nipponbare varieties. Evaluation of 412 markers based on 260 rice varieties resulted in setting up a specific markers asisted for assess the distinctness, the uniformity, and the stability (DUS testing) on rice in our country.
Keywords: DUS (Distinctness, Uniformity, Stability), molecular marker, proprietary variety, rice, test Ngudri phan bidn: GS. TSKH. Trin Duy Quy
