IXIGHIEIV CIJU D A DAIVG D l T R U Y E I V C U A CAC GldlXlii H O A L I L Y , L O A KEIV [LUium ssp.J BAIMG C H i T H f
PHAIXI T U R A P D
Trinh Khac QuangS Nguygn TTii Thanh TTiuf, Bui TTii TTiu Huomg^ v l CTV T6MTAT
Da dang di truyen ciia 13 giong boa lily nhap noi va 3 giong loa ken trong nudc dugc danh gia bing chi tbi phan tii RAPD. 50 moi RAPD da dugc sd dung de danh gia da dang di truyen cua 16 giong hoa ngbien cuu, trong do da ebon dugc 23 moi co tinb da binh cao dtmg de phan tich, danh gia chung. Tren ca sd pban tich ket qua cua 23 moi da thu dugc 308 bang da binh (trung binh 13 bang da hinh/ moi) trong tong so 318 bang (14 bang/ moi). So bang ciia m6i moi dao dong tu 4 den 23 bang, trong do so alen da hinh cua m6i moi la 2 den 23 bang, tuong ung 96,9%. Tren ca sd phan tich khoang each di truyen cho thay do tuong dong di truyen giua cac giong boa nghien cu'u dao dong tu 43 den 82%. 16 giong boa lily va loa ken da pban thanh 3 nhom chinh d do tuang dong di truyen 60%, phii bop vdi phan loai theo dac diem hinh thai va nguon goc cac giong.
Tu khoa: ADN da hinh phong dqi ngdu nhiin, da dqng di truyen, lily.
LDATVANDE
Lily thudc hg Lilium, gom khoang 100 loli phin bd nhieu noi tren luc dia Au-A v l Bac My. Phan Idn cac loli lily cd nguon gdc tu Ddng Nam A (Nhat Ban, Trung Quoc, Hin Quoc), so khic den tif Bac My, cdn lai la tir chau Au (Woodcock & Steam, 1950) [15].
He gen cua lily 11 mdt trong nhung he gen ldn nhat trong cac loli thuc vat. Stt khic biet ve kich thudc he gen giua cic loli lily cung rat ldn (Siljak-Yakovlev & cs., 2003) [11]. Vi du, kich tiiudc ADN cua bd gen (genom) cua L henryi gom 32 trieu cap co sd (base), trong khi mdt so loai khac con so nly cd the len den 100 trieu cap CO sd (base) (Bennet & Smith, 1976; Sentry & Smyth, 1989) [2], [10].
CIc ket q u i nghien cun cho thay cic ky thuat ADN da mang lai do chinh xac cao hon so vdi nhung phuong phip phan loai b i n g hinh fhli trudc day v l dieu nay trg giup cho cic n h i khoa hgc phan loai mdt cich chinh xic hon cic ddng/giong lily (Persson & cs., 1998; Wen & Hsiao, 2001; Horning &
cs., 2003) [9], [14], [5]. Xap xi 100 loli lily da tao ra su da dang di truyen ldn bieu hien qua hinh thii, kich thudc, cic dac tinh sinh trudng...
Cho den nay da cd nhieu chi thi sinh hoa vl chi thi phan tft dugc stt dung de x i c dinh su da dang di truyen thuc vat. Trong so dd, ky thult RAPD (ADN
' Vien Nghien ciiu Rau qua, "TS. Vien Di truyen Nong nghiep, 3 Trudng Dai hoc Nong nghiep Ha Noi
da hinh phdng dai nglu nhien) da dugc su dung de phin tich da dang di truyen cua so lugng ldn cic gidng ciy trong (Galderisi & cs., 1999; Palo mbi &
Damiano, 2002; Al-Khalifah & Asakari, 2003) [4], [8], [1].
Trong khudn khd cua de fai «Nghien cm iing dung cdng nghe sinh hoc tien tie'n cua Hd Lan trong chon, tqo, nhdn gidng vd dieu khien ra hoa chi lilium (lily, loa ken) & Viet Nam", da su dung ky thult RAPD (Random Amplified Polymorphic DNA) de xic dinh da dang di truyen giua 16 gidng lily v l loa ken.
Nhtfng thdng tin ve da dang di truyen thu dugc tif nghien cfm nly phuc vu true tiep cho viec bio ton ngudn gen v l x i c dinh nhtfng to hgp lai trong chgn, tao gidng hoa lily d Viet Nam.
II. VAT LIEU VA PHUDNG PHAP niGHIEN CUU
l.VltUeu
Gidng nhap ndi: 13 gidng hoa lily v l 2 gidng loa ken nhap ngi (Bang 1).
Gidng dia phuong: 1 gidng loa ken (BIng 1).
50 mdi RAPD thudc cac bd kit A, B, C, D, Q, V, W (Operon Tech. Inc., USA) da dugc su dung de danh gil da dang di t'uyen.
2. Phuong phip
Phuang phdp tdch chiet ADN: ADN cua lily va loa ken dugc tach theo phuong phap CTAB cua Doyle v l Doyle ( Doyle, J. J. and J. L Doyle, 1987) [3] cd cai tien.
TT 1
2 3 4
5
6
7 8
9
10
11
12
13
14
15
16
Bang 1. Ttn, ky hitu vk dac di^n:
Ten gidng BELLADONNA BRUNELLO CEBDAZZLE
Lilium formolongo (loa ken)
GOLD CITY
Ll
Lilium longifonim (loa ken) gidng dia phuong
MANISSA
PALMARES
SACRE COEUR Qoa ken)
SIMPLON
SORBONNE
TIBER
VALPARAISO
VENTIMIGLL\
YELLOWEEN
Ky hieu BEL BRU CEB FOR
GOLD
Ll
LONG MAN
PAL
SAC
SIM
SOR
TIB
VAL
VEN
YEL
I gidng Uiy, loa ken su dung trong nghi&n cuu Dac diem cua gidng
Than cung, mlu xanh dam. Hoa mau vang, phan canh dii, cic hoa bd tri hgp ly tren cinh, hoa quay ngang Than cung, mau xanh dam. Hoa mlu vIng sing, phan canh, cudng cic hoa mgc sat nhau, hoa hudng len tren Than cung, mau xanh nhat. Hoa mau cam, mgc thanh chum, cudng hoa dai, hoa hudng len tren
Than cung, mlu xanh nhat. La dii, to. Hoa mau trIng, cudng hoa dii, hoa hudng len tren, cd mui thom nhe Than rat cung, mlu xanh dam. Hoa mau vang, vien frIng, mgc fhlnh chum, cudng hoa dai, hoa rat to, cinh hoa dly, hoa quay ngang
Than cung, mau tim nhat. Hoa mau vang mgc thanh chum, cudng hoa dai, hoa hudng len tren (gin gidng Yelleween)
Than cung, mau xanh nhat. La dai, nhd. Hoa mau trIng, quay ngang, cudng hoa dai.
Than cung, mau xanh dam. Hoa mau vang, phin cinh ngln, cudng hoa dai, hoa quay ngang
Than cung, mlu xanh nhat. Hoa mlu vIng, cd cham do, phan canh ngln, gan nhu mgc thanh chum tren ngon, cudng hoa dai, hoa quay ngang
Than cung, mau xanh. La dai, nhd. Hoa mau trIng, hudng len tren, cudng hoa dai.
Than mem, nhd, mlu xanh nhat, ddt than ngln. Hoa mlu trang, mgc thanh chum, cudng hoa ngln, hoa hudng len tren.
Than cung, mau xanh nhat. Hoa mlu hong, cd cham dd, vien trIng phan canh rat dai, hoa hudng len tren, cinh hoa nhgn va hoi cong ve phia sau, mep hoa lugn sdng.
Thin Cling, gdc thin cd mau tim. Hoa mlu hdng dam, co cham dd, •vien trIng, phan cInh dii trung binh, hoa quay ngang, canh hoa trdn, blu.
Than rat ciing, mlu xanh nhat. Hoa mlu vang, phan canh dai, cac hoa bd tri hgp ly tren canh, hoa hudng len tren, mep hoa lugn sdng rat rd, canh hoa cong gap ve phia sau
Than rat cung, mlu xanh dam. Hoa mau vang, vi^n trIng mgc thinh chum, cudng hoa dii, hoa rat to, canh hoa day, hoa quay ngang.
Than cung, mau tim nhat. Hoa mau vang mgc thinh chum, cudng hoa dai, hoa hudng len tren.
Ky thudt RAPD-PCR: Phan ting PCR (tinh man cam bi ddng cua mIng) vdi mdi RAPD dugc tie'n hinh trong the tich 15 pi dung dich bao gdm:lX Buffer, 0,2 mM dNTPs, 2,5 mM MgCl2, 1 mM Primer, lU Taq vk 50ng ADN. Trong dd cd su cii tien de lam tang do nhay cua phan ting bang each bd sung PVP 10% vl BSA 10%. Phan ting dugc chay vdi 45 chu trinh nhiet 93°C - 1 phut, 36»C - 40 giay, 72''C - 2 phut, bien tinh a trong 5 phut va tdng hgp d budc cudi 72°C - 4 phut.
Phuang phdp phdn tich da hinh di truyen: Cac bang ADN dugc nhap vio chuong trinh Excel theo quy tic: Hien bang dinh sd 1, khdng hien bang danh sd 0. Sd lieu nghien cun dugc phan tich bing chuong trinh NTSYS pc2.1 de xay dung ma tran tuong ddng bieu hien cho mdi quan he gan xa ve mat di truyen vl so dd hinh cay bieu didn mdi lien ket di truyen giiia cac mau nghien cun.
III. KET QUA VA THAO LUAni
1. Nhan dang ADN bang cac chi thi phan tu RAPD
Trong tdng sd 50 mdi RAPD dugc sft dung de sing Igc cd 23 mdi cho ket q u i da hinh tdf (bing 2), sd mdi nly da dugc sft dung de phan tich cho toan bd 16 gidng lily va loa ken nghien cun. 27 mdi cdn Iai cho bang" it, khdng da hinh, bang hi md hoac khdng cho sin pham PCR. Ket q u i phan tich da hinh cua cic mdi RAPD dugc tdng hgp trong b i n g 2. Tdng sd bang thu dugc tft 23 mdi II 318 bang, trong dd cd 308 bang cho da hinh, chiem 96,9%. Mdi mdi cd sd bang tu 4 den 23 bang va sd bang da hinh/ mdi dat 2 den 23 bang, trung binh 11 13 bang da hinh/mdi. 14/23 mdi nghien cun cho 100% bang da hinh. Hinh 1, 2 II Inh chat gen minh hoa ket qua phan tich da hinh ADN giua 16 gidng hoa nghien ctru vdi hai mdi OPB- 04 vl BIO-16.
Cho den nay, sd cdng trinh nghien cun he gen Lilium su dung chi thi phan tir RAPD khdng nhidu.
Lee et al. (1996) [6] da chi su' dtmg 3 mdi RAPD de phan biet 23 gidng Lilium. T i e gil da nhan thay muc do da hinh rat cao, vdi trung binh 17 bang da hinh/
mdi. Yamagishi (1995) [16] da phan biet 37 ddng, gidng thuin, gidng Iai vl c l the Lilium sft dung 9 mdi RAPD. Trong nghien cfm cua Persson et al. (1998) [9] cung da su dung chi fhi RAPD de dinh gia khic biet di truyen vl mdi quan he gitfa 16 quin the Lilium martugon nhap ndi vl b i n dia. Trong sd 25
mdi dugc sing Igc, thu dugc 6 mdi cho bang da hinh.
Ket qui phan tich 6 mdi nly da thu dugc 55 bang da hinh, mdi mdi cho tu 3 - 15 bang da hinh, trung binh 9 bang da hinh/mdi. Khi so sinh ket qua vdi cac cdng trinh nghien cun da cdng bd, da nhan thiy sd bang da hinh thu dugc trong nghien cifu nly 11 kha cao, mdi mdi cho fu 2 - 23 bang da hinh, trung binh
13 bang da hinh /mdi.
BIng 2. K^t qua phin tich da htnh cua cic moi RAPD nghifen ctru
STT
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Ten moi
A8 A9 C5
cn
D3 D5 Q5 Q6 VIO BIO-07 BIO-08 BIO-16 0PA12 OPAJ04 OPB-01 OPB-04 OPB-05 OPB-07 OPW-04 OPW-08 OPW-12 OPW-13 OPW-18 Tong so
Trinh tu
S'-GTGACCTAG -3' 5'-GGGTAACGC -3' 5'-GATGACCGC -3' 5'-AAAGCTGCG-3' 5'-GTCGCCGTC A-3' 5'-TAGGCGGAC A-3' 5'-CCGCGTCTlG-3' 5'-GAGCGCClT G-3' 5'-GGACTTGCT G-3' 5'-GGTTCGCTCC-3' 5'-GGACTCGA(n-3' 5'-TCGAGACGGA-3' 5'-TCGGCGATAG-3' 5'-GAATGCGACC-3' 5'-GTriCGCTCC-3' 5'-GGACTGGAGT-3' 5'-TGCGCCCTrC-3' 5'-GGTGACGCAG-3' 5'-CAGAAGCGGA-3' 5'-GACTGCCTCT-3' 5'-TGGGCAGAAG-3' S'-CACCCGGAA C-3' 5'-TTCAGGGCAC-3'
Tong so bang
15 23 19 19 17 15 15 14 19 9 18 10 10 16 4 12 10 11 17 8 12 12 13 318
Sd bang
da hinh
15 23 19 19 17 15 15 11 19 9 18 10 8 16
2 12
9 11 17 6 12 12 13 308
% bang
da hinh
100 100 100 100 100 100 100 78 100 100 100 100 80 100
50 100
90 100 100 75 100 100 100 96,9
Hinh 1. Sin pham PCR cua 16 gidng hoa vdi chi thi moi OPB-04
Hinh 2. San phim PCR ciia 16 gidng hoa vdi chi thi moi BIO-16
2. Phin tich da dang di truydn giira cic dong/gidng hoa lily vl hoa loa ken
Ket qui phan tich tir ma ti-ln tirong ddng di truyen (bang 3) cho tiiay, dd tuong ddng di truyen giua cac cap gidng nam ti-ong khoang tif 0,43 den 0,82. Cap gidng xa nhau nhat ve mat di truyen II FOR vl Ll vl cap gidng gin nhau nhlt ve mat di ti-uyen 11 VEN vl VAL.
Hinh 3. So do hinh ciy bilu ditn mdi htn k^t di truyin gitra cic gidng hoa nghi6n ctru
So dd hinh cay (Hmh 3) mmh hoa mdi lien ket di truyen gitia 16 gidng hoa nghien cun. 6 muc do tuong ddng di truyen 60% thi 16 gidng hoa da phan Ilm 3 nhdm chinh:
Nhdm I gdm 11 gidng: BEL, PAL, GOLD, MAN, Ll, YEL, VAL, VEN, SIM, SOR, TIB. Khi xet d khoing cich di truyen khoang 63%, nhdm nly da tich fhlnh 3 phin nhdm rd ret. Qua phan tich cho thay, cic gidng trong ffmg phan nhdm cd ngudn gdc vl mdt sd chi thi hinh fhli gidng nhau:
Phdn nhom 1: BEL, PAL, GOLD, MAN II cic gidng thudc nhdm lily OT (la con lai giu'a lily Orient vk Trumpet) vk cd vi tri ra hoa quay theo phuong thing dung.
Phdn nhom 2: Ll, YEL, VAL, VEN la cac gidng thudc nhdm lily OT (11 con Iai giua lily Orient vl Trumpet) vk cd -vi fri ra hoa quay theo phuong ngang.
Phdn nhdm 3: SIM, SOR, TIB II nhung gidng lily thudc nhdm Iai Phuong Ddng (Oriental Hybrids).
Nhdm II gdm 2 gidng lily 11 BRU vl CEB, dly la hai gidng thudc nhdm LA Gl con lai giu'a Longifonim vl nhdm lai ^stfl).
Nhdm III gdm 3 gidng loa ken: FOR, SAC, LONG. Ba gidng loa ken nly da tich thanh nhdm rieng biet vdi cic gidng lily nghien ctru.
Nhung ket qui thu dugc cho thay da cd su tuong quan giu'a cic ket qui phan tich bing chi thi phan tir RAPD vdi mdt sd dac diem ve ngudn gdc vl hinh thai cua cic gidng hoa nghien cun.
BEL BRU CEB FOR GOLD Ll LONG MAN PAL SAC SIM SOR TIB VAL VEN YEL
BEL 0,61 0,55 0,55 0,62 0,58 0,52 0,66 0,69 0,56 0,60 0,61 0,57 0,60 0,61 0,53
Ba
BRU _ 0,70 0,58 0,54 0,57 0,54 0,57 0,59 0,63 0,63 0,59 0,52 0,53 0,58 0,52
ngS.l
CEB
0,59 0,52 0,52 0,57 0,53 0,52 0,60 0,57 0,55 0,55 0,51 0,60 0,52
Via trin tuong dong di truyin giiia cic gidng lUy vl loa kin nghien ciiu
FOR
0,54 0,43 0,65 0,55 0,52 0,72 0,62 0,58 0,50 0,51 0,53 0,45
GOLD
— 0,68 0,51 0,70 0,59 0,48 0,62 0,60 0,57 0,65 0,68 0,62
Ll
_ 0,48 0,60 0,59 0,48 0,55 0,59 0,59 0,66 0,68 0,75
LONG
0,50 0,49 0,69 0,54 0,51 0,51 0,54 0,57 0,48
MAN
0,70 0,55 0,67 0,67 0,54 0,69 0,65 0,49
PAL
_ 0,54 0,64 0,66 0,60 0,60 0,64 0,52
SAC
— 0,62 0,59 0,50 0,52 0,53 0,44
SIM
— 0,81 0,68 0,66 d,65 0,56
SOR
-
^ 6 9 0,69 0,65 0,55
TIB
- 0,65 0,68 0,61
VAL
- Q,8_2 0,63
VEN
- 0,74
YEL
-
Tu cle ket qui phin tich fren da tien hinh phan tich chgn cic cap Iai thich hgp cho cdng tic tao gidng. CIc cap Iai dugc chgn cd do tuong ddng di truyen nlm frong khoing 60-75% vdi muc dich chgn dugc nhifng cap Iai cd the mang Iai uu the Iai cao vl the he con lai cd khi nang sinh sin tdt (bing 4).
BIng 4. CIc cap lai dugc chgn tii kit qua phin tich da (
STT 1 2 3 4 5 6 7 8 9 10 11 12 13
ang di truyin bi Cap lai BRU X CEB BRU x SIM BRU X SAC MAN X BEL
MANxLl MANX VAL MAN X SIM MAN X SOR MANX PAL LONG X SAC LONG X FOR FORX SAC FOR X SIM
Ing chi thi STT
14 15 16 17 18 19 20 21 22 23 24 25 26
phin tii RAPD Cap lai TIB X VEN TIB X SOR TIB X SIM VALxLl VAL X SOR VENxLl VEN X GOLD VEN X YEL YEL X VAL YELxLl GOLD x Ll PAL X BEL SAC X SIM IV. KET LUAN
Su dung ky thult RAPD tai 23 dia diem Ooci) de xic dinh da dang di truyen cua 16 gidng hoa lily vl loa ken nhap ndi vl gidng dia phuong cho thay:
23 mdi RAPD da tao 308 bang da hinh giua cic gidng hoa. Ma tran tuong ddng di truyen vl so dd hinh ciy dugc thief lap tren co sd cle bang da hinh.
Dd tuong ddng di truyen gitra cic gidng tir 43 den 82%. O muc do tuong ddng di truyen 60% thi 16 gidng hoa da phin thinh 3 nhdm chinh phu hgp vdi phin loai hinh thai vl ngudn gdc.
Tren eg sd dinh gil vl so sinh khoing cich di tinyen da xic dinh dugc 26 cap lai tiem nang.
V. TAI UEU THAM KHAO
(1) AI-Khalifah N. S & Askari E. (2003).
Molecular phylogeny of date palm (Phoenix dactyltfera L) cultivars from Saudi Arabia by DNA fingerprinting. Theor. Appl. Genet. 107: 1266-1270.
(2) Bennet M. D. & Smith J. B (1976). Nuclear DNA amounts in angiosperms. Philos. Trans. R. Soc.
London Ser. B 274: 227-274.
(3) Doyle, J. J. and J. L. Doyle (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19:11_15.
(4) Galderisi U. Cipollaro M. Di Bernardo G. De Masi L. Galano G & Cascino A. (1999). Identification of hazelnut (Corylus avellana) cultivars by RAPD analysis. Plant Cell Rep 18: 652-655.'
(5) Horning M. E, Maloney S. C. & Webster M.
S. (2003). Isolation and characterization of variable microsatellite loci in Lilium philadelphicum (Liliaceae). Moi. Ecol. Notes 3: 412-413.
(6) Lee J. S., Lee P. O. Lim Y. P. Shin E. M. Park S. Y. and Rob M. S. (1996). Classtiication of lihes using random amplified polymorphic DNA (RAPD) analysis. Acta. Horf. 414 Proc. Inf. Sym. on Lilium:
137-144.
(7) Nesbitt;, K. A. , Potts, B. M. , Vaillancourf, R.
E., West, A K., Reid, J. B. / Heredity (1995).
Partitioning and distribution of RAPD variation ui a forest tree species. Eucalyptus globulus (Myrtaceae).
Vol. 74, No. 6, pp. 628-637, 32 ref.
(8) Palombi M. A & Damiano C. (2002).
Comparison between RAPD and SSR molecular markers in detecting genetic variation in kiwifruit {Actinidia deliciosa A Chev). Plant Cell. Rep. 20:1061-1066.
(9) Persson H. A., Lundquist K & Nybom H.
(1998). RAPD analysis of genetic variation within and among populations of Turk's-cap lily {Lilium martagon L) Hereditas 128: 213-220.
(10) Sentiy J. W. & Smyth D. R. (1989). An element with long terminal repeats and its variant arrangements in the genome of Lilium henryi. Moi.
Gen Genet 215: 349-354.
(11) Siljak-Yakovlev S., Peccenini S., Muratovic E., Zoldos v., Robin 0. & Valles J. (2003).
Chromosomal differentiation and genome size in three European mountain Lilium species. Plant Syst.
Evol. 236: 165-173.
(12) Veli- Pekka Pelkonen (2005). Biofechnological approaches in Lily {Lilium) production.
(13) Wachira, F. N. , Waugh, R. , Hackett, C. A. , Powell, W. (1995). Detection of genetic diversify in tea (Camellia sinensis) using RAPD markers.
Genome, Vol. 38, No. 2, pp. 201-210, 41 ref.
(14) Wen C. S. & Hsiao J. Y (2001). Alfitiidinal genetic differentiation and diversity of Taiwan lily {Lilium longiflorum var. formosanum; Liliaceae) using RAPD markers and morphological characters.
Int. J. Plant Sci. 162: 287-297.
(15) Woodcock H. B. D. & Steam W. T. (1950). (17) Sale, M. M. , Potts, B. M. , West, A. K. , Liliesofthe World Counti-y Life Ltd., London. Reid, J. B. (1996). Molecular differentiation within
(16) Yamagishi, M. (1995). Detection of section- ^"^ between Eucalyptus risdonii, E. amygdalina and specific random amplified polymorphic DNA (RAPD) theh hybrids using RAPD makers. Australian Journal markers in Lihum. Theor. Appl. Genet. 91: 830-835. of botany. Vol. 44, No. 5, pp. 559-569, 33 ref.
GENETIC DIVERSITY OF LILIUM GENUS GERMPLASM USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKERS
" Trinh Khac Quang, Nguyen Thi Thanh Thuy, Bui Tlii TTiu Huong et al Summary
Sixteen imported and local lilium varieties -were selected for evaluation of genetic diversity using Random Amplified Polymorphic DNA (RAPD) primers. Fifty primers were screened, twenty-three of which were sbo'wn clear polymorphic pattern and selected to analyze in all of the accessions. A total of 308 polymorphisms bands (an average of thirteen polymorphic band per primer) were obtained. The number of polymorphic bands ranged from 2 to 23, corresponding to 96.9% of the amplification products. High level of genetic similarities was observed in the varieties, ranged between 43% and 82%. Sixteen varieties were clustered in three major groups which were corresponded to taxonomy.
Keywords: Random Amplified Polymorphic DNS (RAPD), genetic diversity, lily.
Ngudi phan hitn: GS. TS. Nguyin Quang Tliach.