ielNom-S6i 01/2020
XAY D L A G B Q CHi THI PHAN T O R A P D \ A I S | DE XAC DINH DO DA HINH VA TAN SO D O T BIEN P NGO
Tran Thj Thuy', Dau Thj Ngpc Ni;j'. N g u u n Ihi 11'-i" • Bui Hong Ngpc'. Tran Hong Quan'. Tran Tbi Nji^-e P K P ' - Tran Dang Khanh', Khiai Hflu Truiiv;. \ ' L-'"t5 ^ ' ' "
TO.M T A T
Nghien cflu da sfl dung chi dii phan tfl RAPD va ISJ de danh gia va x i c dinh t i n sd dpt bien cfla quan the d ngd dupc lao ra tu giy dot bien hat phan vdi hda chil E.MS Ket qua sfl dpng 50 moi RAPD v i 2 moi ISI vdi DNA cua gidng ngo MLIO {giong nen sfl dung de tao quan the dot bien) d i x i c dinh dupc 10 mdi da hinh tfl 3 - 4 bang. Trong 10 mdi sfl dung, cd 5 m6i chay on dinh va lap lai tot gifla 186 mau DNA dUpc tach tfl 186 c i the M l tfl quan the d^t bien. Tan sd ddt bien t n m g binh cho ca 5 mdi l i 1/34,3 kb. Nghien cflu nay la bUdc dau lien danh gii ehit lfldng quan the dpt bien bang ehi thi phan tfl. Kel q u i m d dUdng cho cac nghien cflu khac nhU sang Ipc kieu hinh, giii ma h^ genome, de d i n h gia sau hon quan the dot bien EMS d ngd MLIO l i m co sd cho nghien cflu cd b i n chflc nang gene va chpn giong.
Tfl khda: RAPD. IS), tan so dpi bien I. DAT VAN DE
Cdv ngd la mpt c i y t r d n g cd y nghia q u a n t r p n g t r o n g s i n xuat n d n g nghiep. \ ' i vay, cd rat n h i e u cac n g h i e n cflu tren the gidi sfl d u n g eay ngd lam cay m d h i n h n g h i e n cflu cd b i n v i flng d u n g . Rat n h i e u gene d ngd d i d u p c lam sang td chflc n a n g dfla v i o vice tim va nghien cflu cac bien di tfl n h i e n / n h i n t?o. P h a n Idn cac dot bien nhan tao tren ngd d u p c lao ra b i n g hai each: dflng EMS xfl li hat phan, hoac lai vdi d d n g co t r a n s p o s o n d a n g cd k h i n a n g " n h i y "
{transpose) m a n h (Candela a n d Hake, 2008). Gerald Neuffer d i lao ra rat n h i e u dpt bien dUpc sfl d u n g bdi cac n h i khoa hpc tren the gidi c h o d e n nay (NeutTer, 1994) D e d a n h g i i chat l u p n g cua mdt q u a n the dot bien, ngfldi ta cd the dfla vao tan sd tim t h i y cac ddt bien kieu h i n h k h i c d a n g so vdi d d n g gdc. D a n h g i i nay d e d a n g va trflc q u a n nbUng lai k h d n g t h e p h i t hien c i c dpt bien d i e m k h d n g g i y ra kieu hinh. M vay ngfldi ta se ket h p p vdi p h u o n g p h a p d u n g chi thi p h i n tfl de kiem tra Udc linh tan sd ddt bien.
Mdi R A P D ( r a n d o m amplified pohTnorphic D N A ) la nhflng d o a n nucleotide (oligonucleotide) lObp cd t h e b a m v a o D N A k h u d n va n h a n len s i n p h a m PCR. Sfl xuat hien t h e m b i n g m d i hay mat di m d t b i n g la ket q u i cua viec \ i tri b a m m d i tren D N A k h u d n bi thay ddi it nhat I b p hoac h d n . Trong trfldng h p p gay d p i bien b i n g E.MS, p h a n Idn sfl thay ddi la dot bien d i e m (thay 1 nucleotide). C h e n va c d n g tac vien (2012) d i sfl d u n g R A P D d e fldc tinh tan sd dpt bien tren q u a n the ddt bien lua mi b a n g
EMS. Mdi ISJ ( I n t r o n - e x o n Spliced J u n c t i o n ) l i lo?i mdi dflpc thiet ke dUa tren t r i n h tfl b i o t h u c u a ndi tiep g i i p v i c i t gifla i n t r o n va exon t h e o V/elning va c d n g tac vien (1991); Z e n g va c d n g tac vien {2010).
Mdi ISJ t h u d n g cd 15 nucleotit v i vi t h e nhiet d p n d n g chay T m c u a m d i n i y thfldng c a o h d n {Tm k h o a n g 46''C) m d i R A P D (10 b p T m k h o i n g Sl^C).
Vi vay, kha n a n g lap Iai c u a p h a n flng P C R tdt h d n m o i RAPD. Cach t i n h tan sd d o t bien vdi m d i ISJ gidng vdi mdi I'LAPD.
Bang xfl li hat p h a n vdi E M S tren d d n g ngd npi t h u a n c h u n g MLIO, c h u n g tdi d a t a o dflpc q u a n the dpt bien. Trong n g h i e n cflu nay, c h u n g tdi c h u a n hda dieu kien PCR va s a n g Ipc cac m d i R A P D chay dn d m h d d d n g ngd MLIO, tfl d d x i c d i n h dflpc tan sd dot bien cua q u a n the d o t bien E M S t r e n g i d n g ngd MLIO.
II. VAT LifiU VA PHUONG P H A P N G H I £ N C 0 U 2 . 1 . Vat l i e u n g h i e n cflu
- D d n g m e MLIO cua gidng lai d o n LVNlO dflpc lfla chpn de xfl li dot bien. G i d n g ngo lai d d n LVN10 cd n g u d n gdc cua Vien N g h i e n cflu Ngd, dflpc c d n g n h a n la gidng tien b p ky thuat t h i n g 8 n a m 1994.
Mac dii d i d u p c t a o ra each day 25 n a m nhflng h i e n gidng LVNIO v i n la g i d n g dflpc n h i e u ho n d n g d a n chpn lua vi n a n g suat cao va k h i n i n g thich flng vdi moi vimg sinh t h i i t r o n g ca nUdc.
- C a c mdi R A P D v i ISJ.
' \'ien Di truyen Nong nghiep
STT 1 2 3 ' 4
5 6 7 8 9 10 11 12 13
! '4 15 16 17
T i n moi OPOOl OPO02 OPO03 OPO04 OPO05 OPO06 OPO07 OPO08 OPO09 OPOIO O P O l l 0 P 0 1 2 0 P 0 1 3 0 P 0 1 4 O P 0 1 5 0 P 0 1 6 0 P 0 1 7
Bang 1. D:
Trinh tii mdi ( 5 ' - 3 ' ) G G C A C G T A A ACG TAG C G T CTG T T G CTA AAG T C C G C T C C C ACT CAC CCA CGG GAA CAG CAC TGA C C T CCA GTG T C C CAC GCA TCA GAG C G C CAC AGG AGG C A G T G C T G T GTC AGA GTG AGC ATG G C T TGG C G T CCT TCG GCG G T T GGC TTA TGC
mh sach cac moi
STT 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Ten moi 0 P 0 1 8 0 P 0 1 9 OPO20 OPNOl O P N 02 OPN03 OPN04 OPN05 OPN06 O P N 0 7 OPN08 OPN09 OPNIO O P N l l OPN12 0 P N 1 3 OPN14
RAPD sil dung trong T r i n h tti moi
( 5 ' . 3') CTC GCT ATC G G T G C A C G T ACA CAC GCT CTC ACG T T G ACC AGG G G C G G T ACT CCC GAC CGA CCC ACT GAA C G C GAG ACG CAC CAG C C C AGA ACC TCA GCT TGC CGG C T T ACA ACT CGG TCG CCG CAA CAC AGA CAC AGC GTC ACT TCG TGC GGG
nghien ciiu STT Ten moi
35 36 37 38 39 40 41 -1-
^i 44 45 46 47 48 49 50
OPN15 OPN16 OPN17 OPN18 0 P N 1 9 OPN20 OPFOl OPAA03 OPAA04 OPABOl OPAH03 OPAIOl OPAI05 OPBAOl OPBBOl OPBB05
Trinh tti moi ( 5 ' - 3 - ) CAG CGA C TG AAC, CGA C C T C A T T G G G G A GGT GAG GTC GTC CGT ACT GGT GCT CCG A C G G A I H I G TTA GCG CCC C AGG ACT GCT C CCG TCG GTA G G G T TAC TGC C ACGGGTCACi A CAG C G T TGC C T T C CCC Ac:c C ACA CTG G C T G AACAGGGCCG
Bang 2. Danh sich cic moi ISJ sfl dung trong nghien cflu (trinh lfl moi thiet ke theo Weiningvacpng tic vien, 1991).
STT Ten mdi Trinh tU moi (5*3") 1 E2 GGAATTCCA CGTCCA ! . 2 R2 TGCTGGTTTGCA GGT
2.2. PhUPng phap nghien cflu 2.2.1. PhUdng phdp xti li dot bien
Ngd dupc trdng hai vu d Ha Ndi: vu Xuan trdng vio thing 2. vu Mua trong vao thing 8. Phfldng phap giy dot bien dfldc thflc hien theo {NeufFer (1994). Hat phan ngd dfldc thu thip mdi vad budi sing trong tui bao cd (cd da dflpc bpc tfl chieu hdm trfldc dd). Khoang 1220 ml phan dfldc u vdi 70 ml dju Mineral oil (Sigma) chfla EMS (Sigma) d ndng dp 0,05% trong 30 phut. Sau dd, dung djch hat phan trong dau dflpc nhd vio rau ngd. Cic hat ngd thu dflpc tfl bip xfl li gpi la cac bat M1.
Trong nghien cflu nay sfl dung 186 ca the Ml ngau nhien tfl quan the dpi bien tfl ddng MLIO, 2.2.2. Phuangphdp PCR, dien di
- Mau la dflpc thu thap vi tach chiet ADN long sd theo phUPng phap CTAB cua (Doyle JJ va Dolyl IL, 1990).
- Phan flng PCR dflpc tien hanh tren may Veriti 96 well Thermal cycler. Tdng the tich phan flng la 15 pi, bao gdm: Taq2X Mastermix (NEB): 7,5 pi;
DNA khudn: 3 pi; mdi {5 [iM); 3 |il, H,0: 1,5 pL.
Chu trinh nhiet: 95"C: 5 phut; sau dd lap lai 40 chu ki: 95"C: 30 giiy, 31°C (vdi mdi RAPD) hoac 46''C {vdi mdi ISJ): 1 phut, 68''C: 2 phut va ket ihflc bing 68''C: 10 phut.
- San pham PCR dupc dien di tren gel agarose 1,0%. Gel dflpc nhudm elhidium bromide 0,5 mg/ml vasoi tren may Alpha Imager 1220 (Alpha Innotech, CA, USA).
- Cdng thflc tinh tan sd dot bien dfla vao sd nucleotide d i dflpc sing loe nhd chay RAPD, ISJ nhfl sau: Vdi moi mdi RAPD cd chieu dai la "n", cd sd bang nhin len d ddng wild-type (chfla giy dot bien) la "X", sd bang mdi xuat hien them (hoac bdt) li "Y", va chay kiem tra "Z" ca the. Tan so ddt bien = Y/((nx2) X X X Z). Don vj 1/base pair (1/bp) theo Chen va cdng tie vien (2012).
2.3. Thdi gian va dia diem nghien cflu
Nghien cflu dfldc thflc hien tfl thing 1/2016 den
thing 12/2018 tai Bd mdn Ky thuit Di truyen, Vien
Dl truyen Ndng nghiep.
Tap chi Knoj P'OC C6nj nan.-
SdUU0)/2020
lILKtTt^LA\ATHAOLL.\N 50 nidi RAPD di dupe su dyng 3.1. Ket qua chuan hoa PCR mdi RAPD va ISJ giong ngd MLIO (gidng nen ..u dunu '^M'
^ ng5 dot bien) va cho ket qui nhu hinh 1
pNA ^n-' u.in iht
Hinh 1. Kel qua chay kiem tra eae moi RAPD cho mau ngd MLlO
Ghi chu: M Id thang DNA O'Generuler Ikb cua Viemw: (-) Id doi chiing dm (co moi 0PNI6) nhUng thay the DNA Uiiiihi bdngnUdc ml. (+) ddi chiing duong: m6iOPNl6. DNA MLIO.
Nhim lfla chpn cic mdi cd nhieu diem bim de phuc vy kiem tra duov nhieu vi tri trong he gene cua ngd, chflng tdi chpn nhflng mdi cd len tfl ba bing trd len de chay lai vi cho kel qua nhu hinh 2. Cac mdi RAPD cho lfl 3 bang trd len va dn dinh qua hai lan lap lai li: 0 P 0 3 , 0 P 0 6 . 0 P 0 7 , OPOIO. OPOl 1.
0P012. OPN3, 0PN4, OPNll, 0PN13, OPN16, OPNIS. OPFOl. OPAA03, OPAA04. OPABOl,
OPAH03, OPBAOl, OPBBOl (Hinh 2). Trong sd cic mdi RAPD chay dn dinh nay, cac mdi cd sd bang rd vi cd tfl 4 bang trd len Ii tim moi: 0PN4, OPNl 1, OPN16, OPN18, OPAA03, OPABOl, OPBAOl, OPAH03. Hai mdi ISJ chay kha dn dinh la E2 va R2.
Chung tdi se flu tien sfl dung 10 moi nay trfldc de sang Ipc quan the dot bien.
Hinh 2. Ket qui chay l^p lai cic mdi RAPD vdi DNA cua gidng ngd MLlO Ghi chu: M (1) thang DNA O'Generuler Ikb cua Thermo.
3.2. Xac dinh tan so dot bien b^ng chi thi phan tfl RAPD va ISI
3.2.1. Tdn so dot bien tren dongMLlO Trong 10 mdi sfl dung, xic dinh dupc 5 mdi chay dn dinh va lap lai tdt tren 186 mau ei the .Ml lu
quan the dpt bien. Cic mdi khac phan flng PCR len
kem hoac khdng cd dp lip lai dn dinh giOa cac mau
DNA nen chflng ldi khdng sfl dung kel qua de tinh
tan sd dpt bien. Ket qua dupc tdm til trong bine 2
vi hinh 3. Chung tdi chi sfl dung cac bang nio rd
lap lai dflpc d cic mau chay de tinh tan so dot hien'
Vi d p : m d i R2 c d t h e m m d t sd b i n g kich thfldc tfl 500 - 700 b p nhflng k h d n g rd va k h d n g l i p Iai dfldc d tat c i c i c m a u n e n c h u n g tdi chi t i n h l i c d 3 b a n g rd d ddi chflng {Hinh 3). Ket q u i b a n g 2 c h o thay chi cd 3 m d i ( O P N I 6 , E2, R2) c h u n g tdi p h i t hien d u p c ca th^ c d b a n g d p t bien, h a i m d i O P N l 8 , O P A H 0 3 khong xuat hi?n c i t h e ddt bien. Mdi O P N 1 6 , E2, R2 deu xuat hi?n I vi tri d p t bien (I c i t h e / 1 8 6 ca t h e kiem tra). Tan sd dot bien c u a 3 m d i 0 P N 1 6 , E2, R2 lan Iflpt la 1/26,7 kb; 1/27,9 k b ; 1/16,7 kb. N b u vay, tinh c h u n g c h o ca 5 m d i {tdng sd d o t b i e n ( 3 ) / t 6 n g sd nucleotide d i s i n g lpc tfl ca 5 m o i (103 k b ) : t a n so dpt bien la 1/34,3 kb. Lu v i c p n g t i c vien (2018) dung EMS dpt bien h^t p h a n tren g i d n g ngd B73, sau do d i giai m a exon (exon capture) c i c d d n g M l , tfl dd tinh t r u n g b i n h 4 5 8 5 d p t bien t r e n m p t d d n g M 1 . Vdi h^ g e n o m e ngd la 2,300,000 kb, tan s o d p i bien cua q u a n t h e t r o n g n g h i e n cflu b d i Lu v i c d n g t i e vien (2018) l i 1/500 kb. Tuy n h i e n , day la dfla t r e n giii ma chi d o ? n exon chfl klidng phai g e n o m i c n e n lan sd d p t bien c u a Lu va cac c p n g t i c vien (2018) tren c i h? g e n o m e cd the c a o h P n .
0PN16
H i n h 3. Hinh anh chay di^n di s i n pham PCR cd xuat hien bang dot bien cua cic m6i E2, R2 v i OPN 16
Ghi chu: Mui ten chi mdu co so bdng ihem/bdt do dot bien.
Bang 2. Ket qua x i c d m h tan sd dot bien bang chi thi p h i n tfl
Ten mdi Trinh tU (5'-3')
OPN 16 AAGCGACCT OPN 18 GGTGAGGTC OPAH03 GGTTACTGCC E2 GGAATTCCA CGTCCA R2 T G C T G G T T T G C A G G T
So Nu ctia m o i (n)
9 9 10 15 15
So b a n g PCR ro cr doi chting
(X) 8 5 4 5 3
So h a n g them/bdtt xuat hien (Y)
1 0 0 1 1
S < c i t h £ kiem t r a (Z) 186 186 186 186 186
T i n sd dot bien (F) 1/26,7 kb 0/16,7 kb 0/14,9 kb 1/27,9 kb 1/16,7 kb
d cic loii khic sfl dung cac phfldng phip dot bien khic cho ra cac tan sd dot bien khac nhau. Theo nghien cflu cua Chen va cpng tic vien (2012), sfl dyng phin tich cac doan mdi RAPD va ISJ phat hien tan so dpt bien trong lua mi la 1/34 kb, Uauy vi cdng tac vien (2009) - la 1/49,4 kb va Parry va cdng tac vien (2009) - li 1/49 kb. Nhflng mat dp dot bien cao hdn trong lfla mi sfl dyng cung ndng dp EMS nhfl Slade va cpng tic vien (2005) la 1/24 kb va Dong va cpng tic vien {2009) - la 1/30 kb. Cd nhieu cac yeu td eo the anh hfldng den tan sd dot bien, chang han nhfl ndng dp dot bien, thdi gian tiep xuc, tac ddng mdi trUdng va kieu gen cua loai muc tieu (Song va Henry, 1995).
IV. KET LUAN \A DE NGHI 4 . 1 . K e t l u a n
T a m soat v i x i y dflng bd chi Ihi b a o g d m 50 c h i thi R A P D va 2 chi thi ISJ. C h p n dfldc mfldi mdi chay on d i n h va b a n g rd net tren gidng ngd ndi MLIO la 0 P N 4 , O P N l 1 , O P N I 6 , O P N 1 8 , O P A A 0 3 , OPABOl, O P B A O l , O P A H 0 3 , E2 va R2.
Ket q u a chay 3 chi thi p h a n tfl R A P D ( 0 P N 1 6 , O P N 1 8 va O P A H 0 3 ) va 2 chi thi ISJ (E2, R2) tren 186 ca t h e c u a q u a n the ddt bien c h o thay tan sd dpt bien t r u n g b i n h la 1/34,3 kb.
4 . 2 . D e n g h i
iTng d u n g b d chi thi R A P D de sang lpc n e n di truyen c h o c h p n gidng, d e k i e m tra d a h i n h q u a n the.
De b o s u n g lam rd c h o ket q u i n i y . c h u n g tdi se tien h i n h giai m i trinh tU ca he gene c u a m d t sd ca the d p i bien d e x i c d i n h cu the ed b a o n h i e u dot bien t r o n g mdt ca the.
T A I L I E U T H A M KHAO
Candela H, Hake S., 2(K)8. The art and design of genetic screens: maize. Suture review's Genetics. 9: 192-203.
Chen L, Huang L, Min D, Phillips A, Wang S, Madgwick P | , Parry .MA, Hu VG., 2012 Development and Characterization of a New TILLING Population of C o m m o n Bread Wheat [Triticum aestivum L.).
PLoS ONE. 7 (7): e41570.doi:10.1371/|ournal.pone.
0041570.
D o n g C, Dalton-Morgan I, Vincent K, Sharp P. 2009.
A Modified TILLlNCi Method for Wheat Breeding.
Plant Gen.. 2 39-47
Doyle IJ, Doyle IL, 1990. Isolation of plant DNA from fresh ti.ssue Focus. 12 13-15
Lu, X., Liu, J., Ren, W., Yang, Q., Chai, Z., C h e n , R., Wang, L., Zhao, J., Lang, Z., Wang, H., Fan, Y., Z h a o , ). a n d Z h a n g , C , 2018, Gene-Indexed M u t a t i o n s in M.nze. Moicciilur Plant. 11(3).
p p 496-504
Neuffer M (i. 1994, \iiitti\:cnesis. In: Freehng M , Walbot V. (eds) The Maize Handbook Springer Lab Manuals. Springer. New York. NV, pp 212-219.
Parry M A , Madgwick P). Bayoi K, He r na n de z - L op ez .\. Baudo
Tearall M, H a m a d a \V, Al-Yassin A. O u a b b o u II. 1 at'^iiili M, PhiUips AL. 2009. .Mutation dis.^'^^'^ ^'"' ^"'f' improvement, fournal of F-\penn: • /!.''<""• "'*•
2817-2825.
Slade AJ, Fuerstenberg SI, LoeHler D. Steine .MN.
Facciotti D, 2005. A reverse genetic, nonlransgenic approach to wheat crop improvement b\ TILLING.
Ndt Biotec/iHo/, 23: 75-81.
Song W, Henry RJ, 1995. Molecular analysis of the DNA polymorphism of wild barley {Hordeum sponlaneum) germplasm using the polymerase chain reaction. Genetic Resources and Crop Evolution, 42 273-281.
Uauy C, Paraiso F, C o l a s u o n n o P, T r a n RK, Tsai H , B Steve, C Luca, D Jorge., 2009. A modified TILLING approach lo detect induced mutations in letraploid and hexaploid wheal. BMC plant Biology. 9: 115.
Weining S a n d Langridge P., 1991. Identification and mapping of polymorphisms in cereals based on the polymerase chain reaction. Vteoretical and Applied Genetics. 82 (2): 209-216.
Zeng X, Wang Y, Li W, W a n g C, Liu X. a n d Ji W . 2010. Comparison of the genetic diversity between Triticum aestivum ssp. tibetanum Shao and Tibetan wheat landraces (Triticum aestivum L.) by using intron-splice junction primers. Genetic Resources and Crop Evolution, 57 iS): 1141-1150.
Development of RAPD and ISJ markers for analyzing mutation rate in a Vietnamese maize EMS-induced mutant population
Tran Tin Thuy Dau Thi Ngoc Nga, Nguyen Thi Loan.
Bui Hong Ngoc, Tran Hong Quan, Tran Thi Ngoc Diep, Tran Dang Khanh. Khuat Huu Trung, Vi Lang Son EMS-induced pollen mutagenesis have been used to create a mutant population in a Vietnamese inbred MLIO RAPD and ISl molecular markers were used to evaluate the mutation frequency of Vietnamese maize EMS-induced mutant population. Fifty RAPD and t^vo ISJ markers were used to screen MLIO for robust markers that amplified consistently more than .^ bands across tested samples. Ten such markers were identified and were used to screen a subset population of 186 random individuals .Ml plants. The average mutation frequency was estimated to be 1/34.3 kb. These results are the first step to show thai the mutagenesis was effective allowing other methods such as phenot>'ping and genome sequencing for further evaluation of EMS population in the MLIO.
Keywords: RAPD, ISJ. Mvitjtion frequency, maize mutagenesis, EMS Ngav n h a n b i i : 15/12/2019
N g i y p h i n bien: 9/01/2020
NgUdi p h i n bien: TS. Vu Thj Thu H i e n N g i y duyet d a n g : 13/01/2020