NGHIEN ctru SU DA HINH DI TRUYEN CUA WOT SO
X U A T Xir LOAl M A Y mS6c (Daemonorops poilanei J.
Dransf; B A N G KY THUAT RAPD
Pham Van Dien', Nguyin Minh Thanh', Pham Quang Chung', Nguyin Van Viet' TOM TAT
RAPD (Random Amplified Polymorphism DNA) la ky thuat difa tren PCR (Polymerase Chain reaction) vdi mot moi dcfn co tnnh tff ngau nhien gom 10 nucleotit. Hien nay, RAPD la mot trong so cac ky thuat co do nhay va dp chinh xac cao ttong nghien cthi da dang di truyen cung nhiT trong phan loai. Trong nghien cufu nay. May nifctc (Daemonorops poilanei J. Dransf) ciia 3 xuat xuf la Ha Tinh, Quang Ngai va Nghe An diTdc dung lam nguyen lieu de nghien cUu da dang di truyen. Vdi 10 moi RAPD difdc suf dung, ket qua thu difdc 664 phan doan ADN gom hai loai la: 321 phan doan ADN da hinh va 343 phan doan ADN khong da hinh. Trong do, 5 moi cho ty le da hinh 100%
(0PB4, 0PB13, OPB20, OPC13 va OPC20); 2 moi cho ty le da hinh U-en 50% (OPB6 va OPBll); 2 moi khong cho ty le da hinh (OPB7 va OPC8) va 1 moi cho ty le da hinh thap (OPB9). Tren cd sd cac phan doan ADN da hinh nhan du'dc, cac xuat xU loai May niTdc se diTdc dem phan tich ve moi quan he di truyen difa tren he so dong dang di truyen Jaccard va ma tran UPGMA bang phan mem NTSYS phien ban 2.0. Ke't qua chi ra rang: He so dong dang di truyen Jaccard thu dufdc dao dpng tU 0,51 den 0,91. v
Tff khoa: Mdy nudc, da dgng di truyen, RAPD.
I. DAT VAN DE
May nUdc (Daemonorops poilanei J. DransfJ la cay Uong dem lai gia trj kinh te' cao, vi the' ma cay may tif nhien hien dang dUdc khai thac qua mffc dSn den Uff lffdng bi can kiet, loai nguyen lieu nay ud nen khan hiem va gia thanh cao. Nham gdp phan tai tao lai loai may, vffa de bao ve vffa phat Uien vd'n rffng"hien cd dong tiidi cung ca'p nguon nguyen lieu cho nganh thu cdng my nghe, de tai tie'n hanh phan loai, danh gia mffc do da hinh di Uuyen cua gid'ng May nUdc tai cac xua't xff lam cd sd de Iifa chon dung xua't xff td't phuc vu cho muc tieu neu Uen.
Cling vdi cac dac diem sinh hpc va hinh thai, viec diing chi tiii phan tff Uong nghien cifu phan loai, danh gia mffc do da dang di Uuyen thUc vat cho ke't qua cd do tin cay rat cao. Hien nay, cac ky tiiuat chi tiii phan tff ndi chung va ky tiiuat chi thi phan tff RAPD (Random Amplified Polymorphism DNA) ndi rieng da va dang gdp phan quan Upng Uong viec danh gia da hinh loai may d mUc dp ADN [1, 2, 5, 6, 7]. 6 Viet Nam hien chUa cd cdng trinh nao de cap de'n va'n de da hinh di Uuyen loai May nUdc. Bai vie't neu Ien ke't
'Trffdng Dai hpc LSm nghiep.
qua nghien cffu ve da hinh di tmyen cua mdt sd' xua't xff loai May nffdc (Daemonorops poilanei J. Dransfj bang ky tiiuat RAPD.
IL VAT UEU VA PHl/CfNG PHAP NGHIEN ClJtJ 1. Vat Ueu
Mlu nghien cu^i la la May nffdc, mdi mlu dffdc lay ngSu nhien tff 1 ca tiie Uong mdi mdt khdm may.
Cac khdm may nay dUdc chpn mang tinh dai dien cho tffng xua't xff la: Ha TTnh, Quang Ngai va Nghe An.
Trong do, xua't xff Ha Tinh lay 10 mSu dffdc ky hieu tff DHTl de'n DHT9; xua't xff Quang Ngai lay 7 mlu dffdc ky hieu tff DQNl de'n DQN7; xua't xff Nghe An lay 3 mSu dffdc ky hieu tff DNAl de'n DNA3.
Cac Oligonucleotit (moi RAPD) ed nguon gd'c ciia Operon Technologies Inc. (My), cac hoa chat diing Uong tach chiet ADN td'ng sd' va nhan PCR - RAPD ngudn gd'c tff hang Sigma va InviUogen.
2. PhUdng phap
Tach chiet ADN td'ng so' tff cac mlu la May nffdc theo phffdng phap CTAB (Cetyl Trimethylen Amonium Bromide) cua Saghai Maroof va dong tac gia (1984) [4] cd cai tie'n.
Xac dinh nong dp ADN bang phffong phs^: 4- do
ha'p phu tff ngoai d bffdc sdng 260 nm; kiem Ua ADN Uen gel agaroza 0,8% va quan sat dUdi anh sang tff ngoai, chup anh d may Geldoc.
Ky tiiuat PCR vdi cac moi RAPD (Williams et al, 1990) [8] dUdc tie'n hanh vdi td'ng the tich la 25 l/mSu gom nhffng thanh phan sau: 25 ng ADN khudn, moi RAPD (lOng), dNTP (2,5 mM), MgCI, (50 mM), enzym Taq polymeraja (0,5 ddn vi) va dem tiuch hdp cho enzym. Phan ffng dffdc thffc hien qua 45 chu ky lap lai cua 3 bffdc chinh: bie'n tinh ADN khudn d 94"C - 4 phut, gan moi d 34''C - 1 phut va keo dai chudi d 72"C - 2 phiit. San pha'm cua PCR - RAPD sau dd dffdc dien di Uen gel agaroja 1,2%, nhudm bang bang ethidium bromit va chup anh bang may chuyen dung de phan tich.
Xay dung bieu do hinh cay bieu diin md'i tUdng quan di tmyen giffa cac mau cay May nffdc nghien cffu bang phan mem NTSYS phien ban 2.0 (Rohlf F.
J., 2000) [3].
III. KET QUA VA THAO LUAN
1. Tach chiet va lam sach ADN tong so' tff cac mau cay May nUdc
De tach chiet ADN tdng so' tff cac mau la cay May nffdc, sff dung phffdng phap CTAB cua Saghai Maroof va dong tac gia (1984), la phffdng phap da dffdc dung vdi nhieu ddi tffdng thffc vat khac nhau. Tuy vay khi ap dung qui trinh tach chiet nay, ke't qua thu dffdc .\DN cd chat lffdng khdng td't. Vi vay da thay doi mdt so thanh phan dem tach chiet, cu the la tang nong dp EDTA Uong he dem nham loai Uff hoan toan tac ddng phan cat ADN bdi cac nucleaja ndi bao, tang nong dp NaCl nham ban che sff tao phffc giffa ADN va CTAB, bd sung tiiem polyvinyl pirrolidone (PVP) va sodium dodecyl sulfate (SDS) de lam tang kha nang giai phdng ADN cung nhff tang ke't tiia mdt sd hdp chat polyphenol, polysacharit va protein...
ADN tdng so sau khi thu dffdc, dffdc xac dinh nong dp va kiem Ua dp tinh sach bang phUdng phap do dp ha'p phu tff ngoai d bUdc sdng 260 nm va 280 nm Uen may HewleU Parkard. Ke't qua cho tiia'y chi sd ha'p phu
^260/^280 <3eu ldn hdn 1,8. Dieu nay chUng td san pham ADN tdng sddUdc tach chiet cd dp tinh sach cao. Tiep tiieo chat lUdng ADN tdng sd dUdc kiem Ua Iai bang dien di Uen gel agaroja 0,8% (hinh 1) cho tiia'y ta't ca ADN tdng so cua 20 mEu nghien ciJu deu gpn, tap Uung, khdng dinh gieng cung nhU khdng iSn ARN. Ke't
qua nay mdt ldn nffa khang djnh ADN tdng so tach chiet dffdc tff cac mlu May nffdc hoan toan dap ting cho cdc thi nghiem tie'p tiieo ve nhan PCR - RAPD.
Hinh 1. ADN td'ng so' cua cac mlu May nUdfc
tren gel agaroja 0,8% ' ChU thich: Gieng 1- 9: DHTl - DHT9, gieng 10 -
16: DQNl - DQN7, gieng 17-20: DNAl- DNA3.
2. Phan tich da hinh cac phan doan ADN nhan dU<;c tiif 10 moi RAPD
ADN tdng so cua cac xua't xff loai May nffdc sau khi dffdc tach chiet tiieo phffdng phap CTAB neu Uen dffdc dung Iam khudn cho phan ffng PCR - RAPD.
San pha'm PCR - RAPD tiiu dffdc dffdc dien di Uen gel agaroja 1,2% de kiem Ua sU nhan ngSu nhien cua cac phan doan ADN Uen cac xua't xff nghien cffu.
Vdi 10 mdi RAPD Uen 20 mSu nghien cffu, thu dffdc tdng sd' 664 phan doan ADN gom hai loai la:
321 phan doan ADN da hinh (phan doan ADN chi xua't hien d mau nghien culi nay ma khdng xua't hien d mau nghien cffu kia) va 343 phan doan ADN khdng da hinh (phan doan xua't hien d tat ca cac mSu nghien cffu). Mdt phan ke't qua dffdc the hien Uen hinh 2 va 3. So lieu thu dUdc dUde tdng ke't d bang 1 va bang 2.
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Hmh 2. San pham PCR-RAPD vdi moi 0PC13 tren gel agaroja 1,2%
88 NONG NGHIEP VA PHAT TRIEN NONG THON - SO 8 - THANG 8/2009
Bang 2. Ty Ie ADN da hinh cua cac xua't xff loai may nUdc nghien cffu
Hinh 3. San pham PCR-RAPD vdi moi OPB7 tren gel agaroja 1,2%
Chudiich: 1: DHTl, 2: DHT2,3: DQNl, 4: DHT3, 5:
DHT4,6: DNAl, 7: DHT5,8: DHT6,9: DNA2,10: DHT7, 11: DHT8,12: DHT9,13: DHTIO, 14: DQN2, 15: DQN3, 16:DQN4,17:DQN5,18:DQN6,19:DQN7va20:DNA3.
Bang 1. So' phan doan ADN nhan dU^c cua tffng moi RAPD tren ADN he gen cua cac xua't xff loai May nUdc nghien ciifu
TT
1 2 3 4 5 6 7 8 9 10
Ten moi
0 P B 4 0PB6 OPB7 0PB9 OPBll 0PB13 OPB20 OPC8 0PC13 OPC20 Tong
Tong so'phSn do^in ADN thu
di/^c 14 148 120 61 94 19 11 80 73 44 664
Tong so'phan do^n ADN da
hinh 14 88 0 1 71 19 11 0 73 44 321
Ty ^ % phSn do^n ADN da
hinh 100 59,45
0 1,64 75,53
100 100 0 100 100 48,34
Ke't qua bang 1 the hien so phan doan ADN dUdc nhan len d mdi mdi RAPD Uen tdng so 20 mlu May nUdc d ca 3 xua't xff (Ha Tinh, Quang Ngai va Nghe An). Trong 10 mdi RAPD dffdc sff dung cd 5 mdi cho ty le da hinh 100% (0PB4, OPB13, OPB20, OPCI3 va OPC20); 2 mdi cho ty Ie da hinh Uen 50% (0PB6 va OPB11); 2 mdi khdng cho ty Ie da hinh (OPB7 va 0PC8) va I mdi cho ty Ie da hinh tiia'p (OPB9). Ke't qua nay chffng td sff lUa chpn 10 RAPD Uong nghien cifu la phu hdp dac biet la 5 mdi cbo ty Ie da hinh
100%. Day la nhffng mdi mang Iai hieu qua cao Uong danh gia da dang di truyen cung nhU Uong xac dinh xuat xff loai May nffdc.
TT
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Ten m l u
Dim
Difr2 DHT3 Dm"4 DOTS DHT6 DHT7 DHT8 DHT9 DHTIO
DQNl DQN2 DQN3 DQN4
rxjN5
DQN6 DQN7 DNAl DNA2 DNA3 Tong
Tong so'phan doan ADN nh^n
Avtifc 38 27 34 36 36 30 31 40 37 30 29 34 28 34 27 38 35 30 40 30 664
Tong so phan do^n ADN da
hmh 21
16 17 16 19 11 14 18 20 13 12 17 11 17 10 26 20 13 17 13 321
Ty I? % phan doan da hinh
55,26 59,26 50,00 44,44 52,77 36,66 45,16 45,00 54,05 43,33 41,38 50,00 39,29 50,00 37,04 68,42 57,14 43,33 42,50 43,33 48,34
Ke't qua bang 2 cho bie't so' lffdng vi tri gan mdi RAPD Uen ADN he gen cua tffng mSu May nffdc. He gen cua mau nao thu dffdc cang nhieu phan doan ADN da hinh thi he gen cua mlu dd cang cd sif khac biet ve thanh phan ADN so vdi he gen cua cac mau May nffdc khac. Nhffng mau may nffdc nay se la nhffng nguyen lieu td't cho cdng tac chpn tao gid'ng.
Bang 2 cung cho tha'y, mffc dp da binh di Uuyen May nUdc d 3 xua't xff Ha Tinh, Quang Ngai va Nghe An dao ddng d cac mffc dp khac nhau. Cu the, xua't xff Ha Tinh cd 5 mSu (DHTl, DHT2, DHT3, DHT5 va DHT9) cd ty le da hinh cao hdn mffc da binh tmng binh (48,34%). Tffdng tff, xua't xff Quang Ngai cd 4 mSu (DQN2, DQN4, DQN6 va DQN7) cdn xua't xff Nghe An khdng cd mEu nao. Ve mat ly thuyet, 5 mSu d xua't xff Ha Tinh va 4 mSu d xua't xff Quang Ngai Uen la nhffng mlu cd mffc dp da hinh di truyen cao so vdi mat bang chung tffc la cd y nghia tdt cho chon tao gidng. Tuy nhien, de Iffa chpn dung dude \.3:; .;.j
td't can bie't tiiem ve md'i quan he di tmyen d mffc dp xua't xff va giffa cac ca the trong tffng xua't. De lam sang td van de nay, cac xua't xff loai May nffdc se dffdc phan tich tie'p ve md'i quan he di truydn bang phan mem NTSYS phien ban 2.0.
3. Phan tich moi quan he di truyen giffa cac xua't xff loai May nffdc
Tren cd sd cac phan doan ADN da hinh nhan dffdc d Uen, cac xua't xff loai May nffdc se dffdc dem phan tich ve mdi quan he di Uuyen dUa Uen he so ddng dang di tmyen Jaccard va ma Uan UPGMA bang phan mem NTSYS phien ban 2.0 lam cd sd cho viec lUa chpn xua't xU tdt.
He sd ddng dang di Uuyen Jaccard. cho ta biet mffc dp gidng va khac nhau ve mat di tmyen. Ne'u Ui sd he sd ddng dang di tmyen Jaccard cang gan ve phia sd 0 thi cang khac nhau va ngUdc lai cang gan ve phia sd I thi cang gidng nhau. Ke't qua cho tiia'y he sd Jaccard dao dpng tff 0,51 de'n 0,91. Tren cd sd cd he sd ddng dang di Uuyen, da xay dUng bieu do hinh cay bieu diin md'i quan he di Uuyen giffa cac mEu May nffdc Uong ciing mpt xuat xff va giffa 3 xua't xff May nffdc Ha Tinh, Quang Ngai va Nghe An vdi nhau (hinh 4).
Hinh 4. Bieu dd hinh cay bieu dien moi quan he di truyen cua cac xua't xuT loai may nUdc
Qua hinh 4 cho tha'y, cac mau May nffdc cua 3 xua't xff dffdc chia thanh 2 nhdm ldn. Nhdm 1 cd 2 mEu la DHTl va DQN7 vdi he sd ddng dang di tmyen Jaccard la 0,69. Nhdm 2 gdm 18 mEu cdn Iai va lai dffdc chia thanh 11 phan nhdm nhd vdi he sd ddng dang di Uuyen Jaccard dao ddng tff 0,51 den 0,91. Dac biet 2 mEu DHT7 va DHTIO cd he so ddng dang di truyen cao nha't 0,91. Nhff vay cac mEu May nffdc
trong cung mdt xua't xff khdng dffdc sap xep vao ciing mdt phan nhdm ma chung nam rai rac d cac phan nhdm khac nhau. Dieu nay chi ra 2 dieu: Thff nha't cac mEu May nffdc Uong cung mpt xua't xff cd sff da hinh di Uuyen cao; tiiff 2 la cd sff Ufdng ddng di Uuyen giffa 3 xua't xU Ha llnh, Quang Ngai va Nghe An.
Dieu dd ndi len, khdng cd sU khac biet hay ranh gidi rd rang giffa 3 xua't xff Ha ITnh, Quang Ngai va Nghe An. Ke't qua nay cd tiie do tac ddng cua ye'u td mdi trudng va ye'u to' con ngUdi, van de nay can dUdc nghien ciJu tie'p. 6 day chi tap Uung tim ra nhffng ca the cd mffc dp da hinh di Uuyen cao phuc vu cho cdng tac chpn tao gid'ng. NhU Uen da phan tich 5 mEu:
DHTl, DHT2, DHT3, DHT5 va DHT9 d xua't xff Ha Tinh va 4 mEu: DQN2, DQN4, DQN6 va DQN7 d xua't xff Quang Ngai la nhffng mEu cd mffc dp da hinh di tmyen cao dUdc lUa chpn. Tuy nhien, nhin vao hinh 4 cho tiia'y, mEu DHT3 va DHT5 DHTl va DQN7 dUdc xep vao cung mpt phan nhdm, cd nghia la tffng cap mlu nay cd mffc dp tffdng ddng di tmyen tffdng dffdng nhau, tffc la kha nang Iffa chpn Iam gidng la nhff nhau. Mat khac, cung cho tiia'y 6 mEu DQNl, DHT4, DHT6, DHT8, DHTIO va DNA2 la nhffng mEu tuy cd ty le phan doan da binh khdng cao bang 9 mEu: 5 mEu (DHTl, DHT2, DHT3, DHT5 va DHT9) d xua't xff Ha Tinh va 4 mEu (DQN2, DQN4, DQN6 va DQN7) d xua't xff Quang Ngai, nhUng chung Iai cd mffc dp tffdng ddng di tmyen cao vdi 9 mEu nay bdi chung dffdc xep vao cung phan nhdm vdi 9 mEu neu Uen. Ke't qua nay lam tang them kha nang lUa chpn nguyen heu cho cac nha chpn tao gid'ng may.
IV. KET LUAN
1. Trong 10 mdi RAPD sff dung, chpn ra dffdc 5 mdi la 0PB4, OPB 13, OPB20, OPC13 va OPC20cho ty le da hinh phan doan ADN 100%. Day la nhffng mdi RAPD rat phu hdp cho danh gia da dang di truyen loai May nffdc.
2. Phan tich NTSYS - SIMQUAL cho tha'y, cac xua't xff loai may nffdc cd md'i tffdng ddng di tmyen Uong khoang tff 0,51 de'n 0,91.
3. Cac ca the May nffdc DHTl, DHT3, DHT4, DHT5, DHT6, DHT9, DHTIO d xua't xff Ha Tinh;
DQNl, DQN2, DQN4, DQN6, DQN7 d xuat xff Quang Ngai va DNA2 d xua't xff Nghe An la nhffng ca tiie td't cho cdng tac chpn tao gid'ng May nffdc.
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NONG NGHIEP VA PHAT TRIEN NONG THON - SO 8 - THANG 8/2009TAI LIEU THAM KHAO
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STUDY ON G E N E T I C DIVERSITY OF PROVENCAL R A T T A N SPECIES (Daemonorops poilanei J. Dransf) B Y USING THE R A P D T E C H N I Q U E S
Nguyen Minh Thanh, Pham Van Dien, Pham Quang Chimg, Nguyen Van Viet
Summary
The random amplified polymorphic DNA (RAPD) based on tiie polymerase chain reaction (PCR) witii using a single 10-mer of random nucleotide sequence. RAPD-PCR is one of tiie most sensitive and efficient metiiods currently available for study on genetic diversity and taxonomy. In tiiis study, tiiree provencal Rattan species (Daemonorops poilanei J. Dransf): Ha Tinh, Quang Ngai and Nghe An were investigated by using the RAPD metiiod. By using ten RAPD primers, tiie results obtained toUiI 664 bands out of which 321 bands (48.34%) were polymorphic and 343 bands (51.66%) were monomorphic. Five primers (0PB4, OPB 13, OPB20, 0PCI3 and OPC20) were ratio of 100% polymorphic bands; two primers (OPB6 and OPB 11) were higher tiian 50%, two primers (OPB7 and 0PC8) were not and one primer (OPB9) were lower tiian 50% ratio of polymorphic bands. RAPD data obtained were analysed about genetic correlation by the NTSYSpc ver- sion 2.0 software. The results showed tiiat tiie genetic correlation coefficients oscillated fcrom 0.51 to 0.91.
Key words: Daemonorops poilanei J. Dransf genetic diversity, random amplified polymorphic DNA (RAPD)
NgUdi phan bien: TS. Ha Huy Thinh.
