Hoang Thi Thu Yen vd Dtg Tap chi KHOA HOC & CONG NGHE 120(06): 93-99
NGHIEN CUtJ CHI THI PHAN TU" SSR O MOT SO GIONG/DONG CHE TRONG TAI THAT NGUYEN
Hoang Thi Thu Y^n'*, Le Quang Thiromg', Duong Thi Nhung', Ha Thi Loan^
'Tru&ng Dai hoc Khoa hoc - DH Thai Nguyen 'Truong Tmng hoc Pho thong chuyen Tuyen Quang TOM TAT
PCR-SSR la ky thuat sinh hgc phan ni quan trong, duge sir dung pho bien va hieu qua de nghien ciiu chi thi phan Ui trong chgn gifing, n^ien ciiu da dang di truyen, lap ban do gen, nhan dang di CruySn.. Trong nghien ciiu nay, chiing toi phan tich dac diem kich thude mgt so doan SSR va danh gia su da dang genome cac giong/dong che thu thap tai xa Tan Cuong. thanh pho Thai Nguyen, tinh Thai Nguyen; Cong ty che Song Cau va xa Trai Cai - huyen Dong Hy tinh Thai Nguyen DNA t6ng s6 tir cac mau nghien ciiu duoc su dun^ lam khuon de thuc hien ky thuat PCR- SSR vdi 5 cap m6i dac hieu. Kdt qua khu6ch dai d moi m6i_d6u chi ra su da dang trinh tir lap lai ctia doan SSR va da dang cac phan doan DNA duoc khu^ch dai. Sii dyng ph^n men NTSYS version 2 1 phan tich su da dang cac phan doan DNA duoc khuech dai bang ky thuat PCR-SSR, cac gi6ng/d6ng che nghien ciru duoc chia thanh 2 nhom chinh: Nhom I gom 22 mau che, chia thanh 2 nhom phu. Nhom phu 1 g6m 19 mau (Ml, M2, M17, M7, MIO, M20, M22, M23, Ml I. M21.
M4, M8, M16, M15, M3, M14, M5, M6, M13), he so sai khac giiia chung vdi nhom phu con lai la 0,412 Nhom phu 2 g6m 3 mSu M12. M24 va M25 co he s6 sai khac la 0,252. Nhom chinh 11 gom 3 maulaM9, M18,M19, ba mlu nay CO he so di truyen sai khac ldn nhat so vdi nhom khac la 0.46, Tii khoa: Cay che. Camellia sinensis, da dang di truyen, quan he di truyen. SSR. microsatellite.
PCR-SSR MO DAU
Che la Ihii nudc udng tu nhien lau ddi nhal, duge lieu thu rdng rai vdi ehi phi thap, duge irong chii yeu d cac nude nhiet ddi cua Chau A (An Dg, SriLanka, Trung Quoc, Indonesia, Viet Nam..), Chau Phi (Kenya, Uganda, Malawi..) va Chau Mi la tinh (Argentina).
Cay che cd ten khoa hgc la Camellia sinensis (L) O. Kunlze, thugc ehi che {Camellia), hg die (Theaceae), bd ehe (Theales), Idp nggc lan (Dieotyledonea), nganh nggc lan (Angiospermae) [11]. Theo Wight (1962), ehi die (Camellia) dirge phan loai ihanh 3 loai dd la die Trung Quoc (Camellia sinensis China), An Do (Camellia assamica -Assam) va che trung gian giiia che Trung Qudc va An Dp {Camellia assamica lasiocalyx -Campod).
Ba loai che nay la ngudn goc cho hau het cac loai che trdng d eac nudc tren the gidi hien nay (19]. Theo thong ke ctia Min va dtg (2007) cd khoang 120 loai ehe duge trdng d
* Tel: 0982 75215.1. EmaiTyenhtl@imis edu.v
eae nudc chau A, Trung Qudc ed tdi 97 loai, trong do ehe Camellia sinensis (L) O. Kuntze duge phan thanh 4 thir khae nhau, dd la Ihir che Camellia sinensis var sinensis, Camellia sinensis var pubilimba. Camellia sinensis var Assamica va Camellia sinensis var dehungensis [11]. Dae diem ve gidng va chu ky sdng ciia che phuc tap tao nen mgl sd han ehe cho ehgn lao va eai tien gidng theo phuang phap truyen thdng. Viec phan biel giiia eae thii che thugc che China, Assmam va Compod gap khd khan do tinh chat khong dong nhSl eiia ehe [15]. Han nua, cac md la ve hinh thai khdng phan anh eac bien doi ve mat di iruyen trong cay trdng ma chi cho thay khi md ta ve sinh hoa va sinli ly [17], [18]. Do dd, viec su dung cac chi thi phan lii d miic do DNA duac cac nha khoa hgc quan lam sii dung d6 phai hien cae bifin ddi ve mat di truyen.
Tren lh6 gidi da cd nhieu nghien ctiu da dang cay ehe d miic do phan tii. Genome ehe da duge nghien ciiu bang each su dung eae chi thj phan tii nhu chi Ihj RAPD [7], [8], [16);
93
Hoang Thi Thu Yen vd Dtg Tap chi KHOA HOC & CONG NGHE 120(06): 93-99 ehi thj AFLP [6], [12], [16]; chi thi RFLP
[10]; chi thj SSR [9], [13], [14]. Trong do SSR (simgle sequence repeats - SSR) cdn duge ggi la DNA ve tinh (microsatellite) la mgt doan DNA cd su lap lai cua trinh tu nulceotide nao dd. Hien tugng tdn tai cac SSR Irong CO the sinh vat nhan chuan la kha phd bien. Tuy nhien, tiiy tirng loai ma sd Iugng nucleotide trong mdi don vi lai cd the thay ddi tir mdt den hang true va sd lugng don vi lap lai CO the bien dgng tu 2 den hang ngan lan.
Cac doan lap lai hai, ba, bdn nucletolide phan bd phd bien trgng genome cua cac loai dgng thuc vai. Ky thuat PCR khueeh dai doan SSR (PCR-SSR) vdi mdi Ihiel kg dua Iren Irinh lu genome ciia lirng loai nen chi thi SSR cho kel qua nhanh va cao. Chi thi phan tir SSR vdi nhieu dac tinh nhu dg da dang cao, phan bd rdng rai trong genome, di truyen theo quy luat Mendel, bieu hien ddng hgp irgi. Trinh tu SSR duac xac dinh tir Irinh lu' DNA genome va cDNA (EST - Expression Sequence Tag).
Chi Ihi SSR CO ngudn gdc tii' cDNA/EST (EST-SSR) cd the giiip giam chi phi va thdi gian xay dung chi thi do cac doan cDNA/EST lien quan true tiep den cac gen chiic nang, nen SSR phat trien tir ngudn du Heu nay cd the se hiru ich hon [14],
Nam 2009, nhdm nghien ciiu ciia Sharma da Ihong ke c6 2181 doan EST lir co so dd heu NCBI, Irong dd ed 1223 gen cd trinh tu SSR lir 2-34 nucleotide. Trong 109 gen phan tieh nghien ciiu cd chiia 120 SSR duge xae djnh [14] Theo Ihdng ke cua Ma va edng su (2010), d che eo 6899 doan EST duoc cdng bd tren ngan hang gen va cd Ihem 74 ehi Ihi SSR-EST mdi duge nghien cuu ihuc nghiem [9]. Nam 2012, nhdm tac gia Sahu thong ke duge 12852 doan EST d che, theo linh loan ly thuyat CO 1636 doan EST chiia 2371 SSR.
Khi so sanh cac doan EST g ehe vdi cac gen da biel ehiic nang tu' cae loai khae cho thdy hau hel cac chi thi SSR-EST duge nghien ciiu cho den nay lien quan den cac qua trinh sinh hgc, ihanh phan te bao va ehiic nang phan lir d che [13].
6 Viet Nam, viec ling dung eac ki Ihuat sinh hgc phan tu vao viec danh gia he gen cua cay che trong ehgn tao gidng cay trdng cdn la van dk mdi me. Nam 2004, nhdm tac gia Nguyen Minh Hung, Dinh Thi Phdng da su dung ky thuat RAPD d6 nghien ciiu tinh da hinh ciia mdt so ddng che dot bi6n [2]. Nam 2010, Nguyin Thi Thu Huong va dtg da su dung ky thuat nay de phan tich su da dang trinh lu genome d eac ddng che Shan [3]. Mdt so gidng che trdng d xa Tan Cuang - viing die dac san che ndi tieng eiia tinh Thai Nguyen Cling duge phan tich bang ky thuat RAPD bdi Hoang Thi Thu Yen va dtg [I], [5], Nam 2009, Tran Diic Trung va dtg da nghien ciiu danh gia su da dang di truyen 96 gidng/dong che trdng d Viet Nam bang ky thuat PCR- SSR [4]. Vdi muc dieh nham tim kiem chi thj phan tii cd tiem nang ling dung irong chon gidng che, chiing tdi tien hanh phan lich chi thi SSR d mgl sd gidng/ddng che trdng d cac dia phuang cua tinh Thai Nguyen.
NGUYEN LIEU VA PHUONG PHAP Nguyen lieu
Sii dung la cua mdt sd gidng/ddng ehe duge ihu thap tai xa Tan Cuang - Ihanh pho Thai Nguyen, xa Minh Lap va cong ty che Song Cau thugc huyen Ddng Hy, tinh Thai Nguyen (Bangl).
Phuong phap Tdch chiet DNA long so
Phuang phap lach chiet DNA long so duge thuc Iheo md la cua Hoang Thi Thu Yen va dig [5].
PCR-SSR
Dua Iren ca sd eae dii' lieu mdi SSR da cong bd [9], [14], ehiing ldi thuc hien ky thuat PCR-SSR vdi 5 cap moi d 25 giong/ddng che nghien ethi (bang 2). Phan ung duge Ihuc hien Irong 12,5 )j,l the tich vdi cac thanh phdn:
6,25iil master mix (Qiagen), 0,5 \i.\ mdi F (lOinM), 0,5 \i\ mdi F (lOmM), 1 ^\ DNA (40 ng/^ll); 4,25 pi HjO. Chu Irinh nhiet cua phan ung la: 94''C trong 3 phiil, (94'*C trong 1 phiil,
Hoang Thi Thu Yen vd Dig Tap chi KHOA HOC & CONG NGHE 120(06): 93-99 Ate trong 45 giay, 72''C trong 45 giay) lap
lai 32 chu ky, 72''C u-ong 10 phiit va luu giii d 4"C. San pham PCR-SSR duge bSng dien di iren gel agarose 3% va chup anh bang Gel Doc (Pharmacia Amersham Biotech).
Phdn lich so lieu
Dua vao hinh anh dien di san pham PCR- SSR, sy xuat hien cac bang dien di duge ude lugng kieh thude dua vao marker chuan va thdng ke eac bang dien di vdi tirng mdi d timg mau nghien ciiu. Su xuat hien hay khdng xual hien eac bang dien di duge tap hgp de phan tich so heu Iheo nguyen tac: sd 1- xual hien phan doan DNA va sd 0 - khdng xuat hien
phan doan DNA. Cac sd Ueu nay duge xu ly Iren may tinh Iheo chuang trinh NTSYSpc version pc 2,0 (Applied Biostalistics Inc., USA., 1998) de xac dinh quan he di tmyen cua cac ddng che.
KET QUA VA T H A O LUAN
Phan tich chi thi SSR & cdc mau che nghien cuu b&ng ky thuat PCR-SSR
Ket qua dien di san pham PCR-SSR ciia 5 cap mdi vdi 25 gidng/ddng ehe nghien cuu chi ra su da dang trinh tu SSR. Ket qua dien di san pham PCR-SSR d 3 mdi dae trung nhat duge the hien tren hinh 1, hinh 2 va hinh 3.
Bang I: Cdc mdu che nghien cuu
STT
1 2 3 4 5 6 7 8 9 10 II 12 13
Ky hieu
Ml M2 M3 M4 M5 M6 M7 M8 M9 MIO M i l MI2 M I 3
Ten giong
Keo Am Tich Hi]ng Dinh Bach
T[i777 Shan Phiic Van Tien
LDPI Ttaing Du Yabnkna Long Viin
Bat Tien Yakatamidozi
Kim Tuyen PhuTho 10
Nguon gfic
Cong ty che Song Ciiu
STT
14 15 16 17 18 19 20 21 22 23 24 25
Ky hieu M I 4 M15 M16 MI7 MIS MI9 M20 M2i M22 M23 M24 M25
Ten giong
LDP2 Keo Am Tich Hung Dmh Bach
Kim Tuyen LDP2 Tri 777 Trung Du Trung Du Kim Tuytin Biit Tien
LDPI Tri 777
Nguon goc Cong ly che Song Can
Xii Tiin Cuong
Xa Minh Lap
TT
1
2
3 Ten moi
YS27
YS2S
VTS64 F:5'- R: 5'
R 5' F:5' R- 5'
Bang 2: Thong tin ve cap moi sir dung trong Trinh tir moi
• GGGGATAGTACAAACACACAAC -3' - GCTCCTCTTTCTTCACCACTT - 3' - GTCCCCATTGCTCTTAGTTT - 3' - GACAATCATTGCCACCACAT- 3' ' AGTTGGCTGAATCAGTCCCTT -3' - GCTTAAATCACAATTCAAAGC -3'
ngliien cuu Kieh thuoc
du l<ien 8 0 - l l O b p
170-200 bp
260 -275 bp
Motif lap
GA
(TGKGA 1 TTGTT
Y S 8 3 F, 5'- GAGGATTTGGGTTTGTGAAC - 3 ' R: 5' - TCATTCTCTCTGGCATCACC - 3 ' Y T S 9 8 F: 5 ' - TCACCACCTTCCCTTGATAG - 3'
R: 5' - GCATTGGCAATATGAACATG - 3'
2 5 0 - 6 0 0 bp
2 3 0 - 245 bp
Hoang Thi Thu Yen vd Dtg Tap chi KHOA HOC & CONG NGHE 120(06): 9 3 - 9 9
6 7 1 9 M 10 n 12 13 14 15 16 17 18 M 19 20 21 22 23 24 25
Hinh 1. Hinh dnh dien di sdnphdm PCR-SSR cua 25 mau che vat moi YS27 (M. Marker lOObp, 1 - 25. cdc mdu nghien cuu theo thu tu tai bdng 1)
1 2 3 4 5 6 7 8 9 M 10 11 12 13 14 15 i d 17 18 M 1 9 20 2 1 22 23 24 25
Hinh 2. FTinh dnh dien di sdnphdm (M Marker lOObp, 1 - 25. cdc Vdi mdi YS27, san phSm PCR-SSR du kien cd kich ihudc khoang 80-1 lObp. Ket qua tren hinh 1 cho IhSy san phSm PCR-SSR ciia 25 giong/ddng che nghien ciiu cd kich thude gidng nhu linh toan ly thuyet. Tuy nhien, mdt so mlu cd 2 bang (6, 7, 8, 10, II va 12). Nhu vay, giiia cae gidng/ddng che nghien ciiu cd su da dang alen va ket qua nay phii hgp vdi cdng trinh nghien ciiu da cdng bd [14].
Vdi mdi YS28, san phSm PCR-SSR du ki^n CO kich Ihudc khoang 170-200bp. KSt qua tren hinh 2 cho thay san pham PCR-SSR ciia 25 gidng/ddng che nghien ciiu cd kich thude udc linh klioang 290 - 320 bp. KSt qua chung ldi lliu duac cho thay su sai khac vS kich Ihudc doan DNA so vdi ket qua da duge cdng bd bdi Sharma va dig [14]. IVhu vay, giiia cae giong/ddng che nghien ciiu cd su da dang vS trinh tu (TG)(GA) lap lai. Vdi moi YS83, san pham du kien cd kich thude 250-600bp. San pham PCR-SSR d 25 mau che nghien cuu cd kich thude dao ddng tu 290-320bp. Trgng dd, 96
PCR-SSR ciia 25 mdu che vdi moi YS28 mdu nghien cicu theo thu tu bdng 1)
a kich thude 290 bp chi xuat hien d mau so 9, 18 va 19. Nhu vay moi YS83 th^ hien linh da dang alen khdng cao khi so sanh vdi ket qua ciia Sharma va dig [14].
Vdi mdi YTS64, san phSm PCR-SSR du kign cd kieh Ihudc khoang 260-275bp. Kel qua d hinh 3 cho thay tat ca cac mau deu cd bang khoang 260-275bp, mgl sd mau cd them cac bang phu khoang 300bp (mau 11, 12 va 13) Nhu vay, kel qua vdi cap moi YTS64 cho thay su da dang alen giiia cac mau che khao sal. Vdi moi YTS98, kich thude san pham PCR-SSR du ki8n khoang 230-245bp. Tai vi Iri khoang 240bp tat ea cac giong/ddng deu xual hien bang DNA, hau h6t cae mau co them bang phu khoang dOObp. Nhu vay, kk qua vdi cap mdi YTS98 eho thay hiiu nhu khdng cd su da dang alen giua cac mau che khao sal. Kel qua PCR-SSR mdi YTS64 va YTS98 eiia ehiing tdi gidng vdi ket qua da duge cdng bd nam 2010 bdi Ma va dtg [9].
Hoang Thi Thu Yen vd Dig Tap chi KHOA HOC & CONG NGHE 120(06). 93-99
Hinh 3. Hinh dnh dien di sdn pham PCR-SSR ciia 25 mdu che vdi moi YS64 (M: Marker IQObp, I - 25: Cede mdu nghien cuu theo thic tu bdng 1) Bang 3. Sd phdn doan DNA xudt hien vd sd phdn doan DNA da hinh
doi vai moi moi Moi
YS98 YS28 YS27 YS64 YS83 Tons
Phan l<h
doan DNA ueeh dai
3 4 4 4 3 18
Phan doan DNA da hinh
2 4 4 4 3 17
TV le phi in triiru phan doan da hinh
66,67 100 100 100 100 93.3 Mdi quan he di truyen giira cdc dong che
dira tren phdn tich SSR
Kel qua dien di san pham PCR-SSR cua 25 mau che vdi 5 cap mdi thu duge 18 phan doan DNA, M6i YS27, YS28 va YTS64 c6 4 phan doan DNA duge khuech dai, mdi YS83 va YTS98 CO 3 phan doan DNA duge khufich dai. Nhu vay, su dung ky thuat PCR-SSR vdi 5 cap mdi SSR cho thay su khae nhau trong cau tnic genome ciia 25 mau che. Tinh da hinh duge the hien d su xual hien hay khdng xual hien phan doan DNA khi so sanh giiia cae mau vdi nhau. Thdng qua ket qua phan lich da dang sii dung 5 cap mdi SSR cho thay so phan doan DNA xual hien la khae nhau va the hien tinh da hinh la khac nhau, sd phan doan DNA xual hien va sd phan doan DNA da hinh doi vdi moi mdi duge the hien d bang 3.
Kel qua d bang 3 cho thay cac phan doan duge khuech dai cd kich thude Idioang 80- 600bp. Tron^ dd cac mdi YS28, YS27, YS64, YS83 cho kei qua da hinh cao (100%). Cdn m6i YS98 eho kit qua da hinh thap ban (66.67%)
D\ra tren su xudt hicn hay khdng xuat hien e;lc phan doan DNA ciia cac mau khi phan
tich dien di san pham PCR-SSR, ehiing loi xac dinh duge he so dong dang di Iruyen eiia cac mau che d cap do phan tii. Sd lieu duge phan tich theo chuang trinh NTSYSversion 2.0 (theo quy udc 1= xual hien bang, 0 = khdng xuat hien bang). Kel qua eho thay he sd di truyen dao ddng tir 0,33 - 1 Trong dd mau Ml va M2, mau Ml va M17; mau M2 va M17; mau M20 va M22 cd he sd tuang ddng ldn nhat la 1, cdn mau M4 ed he sd tuang dong nho nhat vdi mau M12 la 0,33. Kel qua phan lich he sd di iruyen giOa cac mau nghien ciiu duge the hien bang so dd hinh cay ciia 25 mau che d hinh 4. Cu the, cay phan loai duge chia thanh hai nhdm ro rang, he sd tuang dong giua hai nhom la 0,54 (tiic 54%). Nhom I gom 22 mau ehe, chia thanh 2 nhom phu Nhom phu 1 gdm 19 mSu (Ml, M2, M17, M7, MIO, M20, M22, M23, M11,M2I, M4, M8, M16, M15, M3, M14, M5, M6. M13), he sd sai khac giiia chiing vdi nhom phu cdn lai la 0,412. Nhdm phu 2 g6m 3 mSu M12, M24 va M25 cd he sd sai khae lii 0,252 Nhdm chinh II gom 3 mau la M9, MI8, MI9, ba mau nay cd he sd di Iruyen sai khac ldn nhat so vdi nhdm khac la 0,46
97
Hoang Thi Thu Yen vo Dtg Tap chi KHOA HOC & CONG NGHE 120(06): 9 3 - 9 9
Hinh 4. Bieu do quan he di truyen (Ml - M25. cdc mdu nghi K E T L U A N
C h u n g toi su d u n g k y thuat P C R - S S R v d i 5 cap mdi d a e hieu da chi ra s u da alen cac d o a n S S R v a q u a n h e di truyen cua 25 giong/ddng che nghien ciiu.
Ldi cdm an: Cong trinh duoc thuc hien vdi kinh phi ciia de tai cap Dai hoc' "Nghien cuu su da dang trong he gen ciia mot so giong che dac sdn trong tai Thdi Nguyen bdng ky thudt SSR" va trang thiet bi ciia Phong thi nghiem sinh hoc, khoa Khoa hoc Su song, Tmdng Dai hoc Khoa hgc, Dai hgc Thai Nguyen; Vien Khoa hgc Su song - Dai hoc Thai Nguyen
T A I L I E U T H A M K H A O 1. Bao Lam (2011), Tan Cuong - Xiing danh de nhat danh tra Thai Nguyen, thainguyen gov.vn.
2. Nguyen Mmh Hiing, Dinh Thi Phong (2004),
"Danh gia tinh da hinh RAPD genome mgt so giong che". Tap chi Cong nghe sinh hoc, 2(2): 109-116 3. Nguyen Thi Thu Hucrng, Nguyen Thi Thu Phucmg, Hoang Van Chung, Hoang Thi Thu Yen (2010), "Budc dau nghien ciiu da dang di tmyen a mgt sd dong che shan {Camellia sinensis var assamica (Mast) Pierre sec. Phamh) bang ky thuat RAPD". Tap chi Khoa hoc vd Cong nghe Thdi Nguyin, 6X3)' 149-157
4. Tran Diic Trung (2009), "Nghien cuu su da dang di truyen cac gidng/ dong che {Camellina sinensis (L.)0 Kuntze) d Viet Nam bSng chi thj hinh thai va chi thi phan tu microsatelHte(SSR)". Luan van Thac sT, Trudng Dai hgc Bach khoa Ha Ngi.
5. Hoang Thj Thu Y^n, Nguyen Van Tuan, Hoang Thi Nga (2012), "Nghien ciiu da dang di
giira cdc dong che nghien cuu cuu theo thu tir bdng 1)
truyin genome mgt sd dong che {Camellia sinensis) trong tai xa Tan Cuang - Thanh pho Thai Nguyen bSng ky thuat RAPD". Tap chi Khoa hgc vd Cdng nghe Thdi Nguyen, 96(8): 139 -143, 6. Ji P Z., Jiang H. B., Huang X Q., Zhang J., Liang M. Z., Wang P, S. (2009), "Genetic diversity of ancient tea gardens and tableland tea gardens from Yunnan Province as revealed by AFLP marker". Yi chuan, ^XO)- 101 - 108.
7. Ll J , Jiang C. J., Wang Z. X (2005),
"RAPD analysis on genetic diversity of the preconcentrated core germplasms of Camellia Sinensis in China". Yi chuan, 27(5). 765 - 771.
[8]. Lin S. Y., Chen I. Z„ Tsai C. M., Chen Y. L.
(2005), "Detection of genetic relaUonship in Taiwan tea variety {Camellia sinensis (L.) 0 Kuntze) with RAPD markers". Journal of the Chinese Society for Horticultural Science, 51(4):
357 - 366.
9. Ma J. Q., Zhou Y. H., Ma C. L., Yao M. Z., Jin J Q , Wang X. C , Chen L. (2010),
"Identification and characterization of 74 novel polymorphic EST-SSR markers in the tea plant.
Camellia sinensis (Theaceae)". American journal of botany, 97 (\ 2): 1 5 3 - 156.
10. Matsumoto S., Takeuchi A., Hayatsu M., Kondo S. (1994), "Molecular cloning of phenylalanine ammonia-lyase cDNA and classification of varieties and cuUivars of lea plants (Camellia sinensis) using the tea PAL cDNA probe ". Theoretical and applied genetics 89(6): 671-675.
11 Mm T., Bartholomew B. (2007). "18 Theaceae". Flora of China 12- 366 - 367.
12 Mishra R. K., Chaudhury S., Ahmad A., Pradhan M , Siddiqi T. O. (2009), "Molecular
Hoang Thi Thu Yen vo Dtg Tap chi KHOA HOC & CONG NGHE 120(06). 93 - 99
analysis of tea clones (Camellia sinensis) usinh Veen-man H, Zonen >JV. Wageningen, The ALFP markers". International Journal of Netherlands 459-493.
Intergrative Biology, 5{2): 1 3 0 - 1 3 5 . 16. Wachira F., Tanaka J., Takeda Y. (2001), 13 Sahu J., Sarmah R., Dehury B., Sarma K., "Genetic variation and differenUation in tea Sahoo S., Sahu M., Barooah M., Modi M. K., Sen {Camellia sinensis) germplasm revealed by RAPD P. (2012), Mining for SSRs and FDMs from and AFLP vanation ". Journal of Horticultural expressed sequence tags of Camellia sinensis. Science Biotechnology 76(5) 557 - 563.
www.bioinfonnation.net 17. wickremasinghe M. R. (1981), "Vanation in 14. Sharma H., Kumar R., Sharma V., Kumar some leaf charatenstics m tea (Camellia sinensis) v., Bhardwaj P., Ahuja P. S., Sharma R, K. and their use in identification of clones". 7>a, 50.
(2009), "Identification and cross-species 183-189.
transferability of 112 novel unigene-derived 18. Wickremasinghe R. T. (1979), Tea In microsatellite markers in tea (Camellia sinensis)". Advances m food research Volume 24. Edited by American journal of botany, 98(6): 133-138. Mark EM, Steward GF. New York: 229-286.
15. Visser T. (1996), Tea, Camellia sinensis (L.) [ 9. Wight W. (1959). "NomenclaUire and 0 . Kuntze in Outline of Perennial Crop Breeding Classification of the Tea Plant". A'li/u/'e, 183: 1726 in the Tropics Edited by Ferwarda FP W. F , - 1728
S U M M A R Y
I N V E S T I G A T I O N S S R M A R K E R S I N S O M E V A R I E T I E S / C L O N E S T E A (CAMELLIA SINENSIS) I N T H A I N G U Y E N
Hoang Thi Thu Yen'", La Quang Thuong^
Duong Thi Nhung', Ha Thi Loan' 'College of Sciences - TNU. ' Tuyen Quang High School PCR-SSR is important molecular technology which used popular and effective to study molecular markers in breeding, genetic diversity studies, gene mapping, genetic identification. . In this shidy, we analyzed the characteristics of the size of some SSR fragments and evaluated genomic diversity of varieties/clones collected in Tan Cuong commune. Thai Nguyen city. Thai Nguyen province. Song Cau tea Company and Minh Cau commune - Dong Hy district, Thai Nguyen province. Total DNA from the samples is used as a template to perform PCR with specific 5-SSR primer pairs Amplification results of primers are indicated SSR fragments sequence diversity and diversity of the DNA fi-agments to be amplified. Using NTSYS version 2 I software to analysis diversity of DNA fragments amplified by PCR - SSR, research varieties/lines tea is divided into two main groups: Group I included 22 tea samples, divided into 2 subgroups Subnoup 1 consists of 1 9 s a m p l e s ( M I , M 2 , M17, M7, MIO, M20, M22, M23, M 1 I , M 2 1 . M 4 , M8, M16, M i 5 , M3, M14, M5, M6, M13), coefficient of variation between them vnth the remaining 0 412. Subgroup 2 consists of three samples M l 2, M24 and M25 have different coefficient is 0.252 Group II consists of 3 samples M9. MIS and M I 9 , three samples have largest hentability difference was 0.46 compared with other groups
Keywords: Tea. Camellisa sinensis, genetics diversity, genetic relationship. SSR. microsatellite.
PCR-SSR
Ngdy nhgn bdi-14/4/2014, Ngdy phdn bien:05/5/2014, Ngdy duvet ddng 09/6/2014 Phan bien khoa hoc: TS. Nguyin Thi Hdi Yin - Tnr&ng Dai hoc Khoa hgc - DHTN
Tel. 11982 752153. Emad yenhtl@lnus edu v
