Tgp chi Cong nghe Sinh hoc 8(3): 331-336, 2010
P H A N T I C H D A D A N G V A Q U A N H E DI T R U Y E N Q U A N T H E T H U Y T U N G (GLYPTOSTROBUS PENSILIS) 6 D A K L A K B A N G CHI THI SSR
Vii Dinh Duy', BCii Thi Tuylt Xuan\ Trin Vinh^, Nguyin Minh Tam' 'Bdo tdng Thien nhien Viet Nam
^Vien Sinh thdi vd Tdi nguyen sinh vdt
^Vien Khoa hoc Ky thuat Nong Ldm nghiep Tdy nguyen TOM T A T
Da dang di truyen va quan he di truyen cua 40 cay Thuy timg (Glyptostrobus pensilis) thu thap d 2 huyen Krong Nang va Ea H'Leo ciia tinh Dak Lak da dugc phan tich dua vao tinh da hinh cua chi thi SSR. 10 trong tong so 21 cap moi SSR kiem tra da dugc sii dung de danh gia mire dp da hinh va moi quan he di truyen giira cac ca the loai Thuy timg. Tdng sd 21 allele da dugc xac dinh, vdi sd allele trung binh cho mot locus la 2,1.
Gia tri PIC (Polymirphic Informafion Content) dao dgng tir 0,008 (mdi Pt 110048) din 0,229 (mdi Pt 15169), trung binh 0,122. Phan tich nhdm diing phuong phap UPGMA tren co sd he so tuong dong di truyen da phan biet 3 nhom chinh. Mot nhom gom tat ca cac ca the thugc khu Bao ton thien nhien Trap K'So. Cac ca the d EaRal dugc tach thanh 2 nhdm, trong do mot nhdm vdi 3 ca the co quan he rat gan nhau, he sd tuong dong di truyen vugt 90%. Mot sd giai phap cho cdng tac bao tdn va phuc hdi loai Thuy tiing cung dugc de cap.
Tir khoa: Bdo ton. da dgng di truyen, Glyptostrobus pensilis, SSR, Thuy timg
MODAU
Cay Thiiy tiing hay cdn ggi la Thdng nude Glyptostrobus pensilis (Staun) K.Koch, thudc hg Hoang dan (Cupressaceae) la loai cay gd quy dang bi de dga Uiyet chiing (Bd Khoa hgc va Cdng nghe, Vien Khoa hgc va Cdng nghe Viet Nam, 2007). Hien tai, loai nay chi phan bd d Dak Lak vdi sd lugng ca the it trong mdt sd manh rimg thir sinh bi phan cat, (Averyanov et al, 2009; Hiep et ai, 2004; Nguyin Hoang NghTa, 2004; Luu, Thomas, 2004). Theo cac sd lieu dieu tra khao sat ciia chiing tdi vao thang 11 nam 2009, sd ca the ciia loai cdn rat it, khoang 300 ca the, tap trung d 2 manh rimg thii' sinh cdn sdt lai, Trap K'So (huyen KrdngNang) va EaRal (huyen EaH'leo).
Thiiy tiing cd re thd, chiu dugc mdi trudng ngap nude nen thudng sdng d viing nui dat thap sinh lay.
Cay Thuy tiing phan bd rgng d Bac ban cau vao ky De til' (Florin, 1963; Averyanov et al, 2009). Loai nay bi suy giam manh vao ky Bang ha, hien nay, chi tdn tai vdi sd lugng it d Ddng Nam Trung Qudc, Trung Viet Nam va Ddng Lao (Faijon, Page, 1999;
Li, Xia, 2005). Thuy tiing la loai sinh san don tinh va thu phan nhd gid. Trong vai thap ky gan day, dudi ap lire cua su tang dan sd va phap trien kinh te nhanh, nai sdng ciia loai nay bi suy giam. Loai cay nay dang bj de dga tuyet chung khdng chi vi cd noi sdng bi hep va sd ca the cdn lai qua it trong mdt sd manh riing thir sinh cdn sdt lai d Dak Lak. Ngoai ra, viec duy tri muc nude cao hang nam bdi cac con dap
giir nude trong nhirng manh rimg nay phuc vu ngudn nude de phat trien cay ca phe ciia viing cung anh hudng rat Idn den kha nang tai sinh ciia loai cay nay.
Nhieu cay da bi chet va khdng xuat hien cay tai sinh trong vai chuc nam gan day. Chat lugng hat kem ciing phd bien d khu vuc nghien ciru. Ve mat thuong mai, gd Thuy tiing chiu dugc mua nang, khdng bj mdi mgt diing de lam nha va lam dd my nghe. Canh, la, qua diing lam thudc cd tac dung chiia phong, khir thap, cam dau.
Thiiy tiing cd gia tri cao ca ve mat khoa hgc lan kinh te, nhu vay viec bao tdn va phuc hdi loai Thuy timg la yeu cau cap thiet dat ra cho cac nha khoa hgc cung nhu nha quan ly. De gdp phan dua ra cac giai phap bao tdn va phuc hdi loai, danh gia mire do da dang di truyen quan the cay Thiiy tiing (Glyptostrobus pensilis) cd y nghTa quan trgng. Mire do da dang di truyen khdng nhirng chi ra kha nang tdn tai cua loai d hien tai va tuong lai, ma cdn chi ra tiem nang tien hoa cua loai. Ngay nay, ky thuat cdng nghe sinh hgc dugc su dung rdng rai, nhanh va c6 hieu qua trong viec danh gia mire do da dang di truyen quan the va loai, dac biet cac loai thdng dang cd nguy co bi dang bi de dga (Goncharrenko et al, 1993; Shea, Fumier, 2002; Ledig et al, 2005; Quach Thi Lien et al, 2004; Nguyen Dire Thanh et al, 2005; Nguyen Hoang NghTa et al, 2007; Nguyen Minh Tam et al, 2009).
Trong bai bao nay, chiing tdi trinh bay ket qua
phan tich 10 cap mdi SSR de danh gia mire do da dang di truyen va mdi quan he di truyen giua cac ca the trong 2 quan the Thuy tiing sdng ty nhien d Dak Lak, va de xuat mgt sd giai phap bao tdn va phuc hdi chiing d Viet Nam.
VAT LIEU VA PHUONG PHAP Vat lieu
Tdng sd 40 mau la hoac vd cay (ddi vdi cay to khdng lay dugc la) dugc thu thap ngau nhien tai khu Bao tdn thien nhien Trap K'So (huyen Krdng Nang)
vdi do cao 750 m so vdi mat bien tga do 13''0r BJc - I08"09' Ddng, va khu Bao ton Thien nhien EaRal (huyen EaH'Leo) vdi do cao 570 m tga do 13"09' Bac - 108''I8' Ddng (Bang I). Trong thdi gian thuc dia, mau dugc bao quan trong tui nilon chira silica gel. Mau dugc ghi sd ciing vdi dac diem sinh hgc ciia cay lay mau. Sau dd mau dugc chuyen ve phdng thi nghiem Sinh hgc phan tir cua Bao tang Thien nhien Viet Nam va dugc bao quan -30°C trong tu lanh sau cho den khi dugc sir dung de phan tich trong phdng thi nghiem. Mau tieu ban tai dia diem thu mau ciing dugc thu thap giup cho cdng tac xac dinh chinh xac ten loai nghien cu'u.
Bang 1. Danh sacb 40 mau Thuy tung (Glyptostrobus pensilis) thu thap d Tr§p K'So (Krong Nang va EaRal (EaH'Leo).
Ky hieu Ngudn goc mlu
TT Ky hieu Nguon goc TT mau
Ky hieu Nguon TT Ky hieu Nguon mlu gdc miu goc 1
2
3
4
5
6
7
8
9
10 E K 1
E K 2
E K 3
E K 4
E K 5
E K 6
E K 7
E K 8
E K 9
EK 10
T r i p K'So- Krdng Nang T r i p K'So- Krong Nang Trap K'So- Krong Nang T r i p K'Sa- Krong Nang T r i p K'So- Krdng Nang T r i p K'so- Krdng Nang T r i p K'So- Krdng Nang T r i p K'So- Krong Nang Trap K'So- Krong Nang T r i p K'So- Krong Nang
11
12
13
14
15
16
17
18
19
20
EK 11
E K 1 2
E K 1 3
E K 1 4
E K 1 5
E K 1 6
E K 1 7
EH 1
EH 2
EH 3
T r i p K'So- Krdng Nang T r i p K'So- Krdng Nang T r i p K'So- Krong Nang T r i p K'So- Krdng Nang T r i p K'So- Krong Nang T r i p K'Sa- Krong Nang T r i p K'Sa- Krong Nang EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo
21
22
23
24
25
26
27
28
29
30 EH 4
EH 5
EH 6
EH 7
EH 8
EH 9
EH 10
EH 11
EH 12
EH 13
EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo
31
32
33
34
35
36
37
38
39
40
EH 14
EH 15
EH 16
EH 17
EH 18
EH 19
EH 20
EH 21
EH 22
EH 23
EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo EaRal - EaH'leo
Phu'ong phap nghien ciru Tdch chiet DNA tong so
Mau dugc tach chiet theo phuong phap CTAB cua (Doyle, Doyle, 1987) cd cai tien cho phii hgp vdi dieu kien Viet Nam. Kiem tra do sach va ham lugng DNA bang do quang phd hap thu ket hgp vdi dien di tren gel agarose 0,8%. DNA tdng sd dugc pha loang diing cho phan img PCR d ndng do 10 ng/pl.
NlidnbdnDNA
The tich mdi phan irng PCR la 25 pi, trong dd chira cac thanh phan gdm dung dich dem Ix PCR;
2,5 mM MgCL; 2 mM dNTPs; 0,5 pmol cho moi mdi xudi hoac ngugc; 50 ng DNA tdng sd va 0,5U Taq polymerase. Qua trinh nhan ban dugc tien hanh tren may Gene amp PCR system 9700 theo chu trinh nhiet sau: (1) Bien tinh ban dau: 94^0 trong 3 phut;
(2) Bign tinh: 94"C trong 1 phut; (3) Bat cap; 55"C trong 1 phut; (4) Keo dai: 72"C trong 1 phut; (5) Lap lai (2) den (4) 40 chu ky; (6) Phan irng ket thuc hoan toan: 72"C trorig 10 phut; (7) GiO san pham d 4"C.
Dien di san pham tren gel Polyacrylamide 5% trong 40 ml dung djch dem IxTAE, nhudm Ethidium bromide va chup anh tren may soi gel CLEARVER.
10 cap mdi SSR da dugc sir dung de danh gia mire 332
Tgp chi Cong nghe Sinh hoc 8(3): 331 -336, 2010 do da dang di truydn va mdi quan he di truyen giiira cac mau nghien ciru (Bang 2).
Phdn tich so lieu
De danh gia da dang di truyen va mdi quan he di truyen giiia cac mau nghien ciru, san pham SSR dugc xac dinh nhu sau: (1) bang xuat hien va (0) khdng xuat hien tren ban gel dien di polacrylamide
5%. Xac dinh he sd tuong ddng di truyen (Jaccard) va gia tri PIC = 1 - (Pij)", trong dd Pij la tan sd ciia allele thir i trong quan the thir j , gia tri tuong quan kieu hinh (r), de lap ra bieu do so sanh he sd tuang ddng di truyen giira 40 mau Thuy tiing d mire do phan tu. Xay dirng cau true hinh cay (UPGMA) tren CO sd khoang each tuong ddng di truyen giira cac mau nghien ciru. Sd lieu dugc xir ly bang chuong trinh NTSYS-pc (Rohlf, 1988).
Bang 2. Trinh tu cac nucleotide ciia 10 cap mdi SSR diing phan tich SSR.
STT Ten moi Primer Trinh tu moi Tac gia
1
2
3
4
5
6
7
8
9
10
Pt15169
Pt 26081
Pt 30204
Pt71936
Pt110048
Pt 87268
PI
P3
P4
P5
F R F R F R F R F R F R F R F R F R F R
5'- CTT GGA TGG AAT AGC AGC C -3' 5'- GGA AGG GCA TTA AGG TCA TTA - 3' 5'- CCC GTA TCC AGA TAT ACT TCC A - 3' 5'- TGG TTT GAT TCA TTC GTT CAT - 3' 5'- TCA TAG CGG AAG ATC CTC TTT - 3' 5'- CGG ATT GAT CCT AAC CAT ACC - 3' 5'- TTC ATT GGA AAT ACA CTA GCC C - 3' 5'- AAA ACC GTA CAT GAG ATT CCC - 3' 5'- TAA GGG GAC TAG AGC AGG CTA - 3' 5'- TTC GAT ATT GAA CCT TGG ACA - 3' 5'- GCC AGG GAA AAT CGT AGG - 3' 5'- AGA AGA TTA GAC ATC CAA CCC - 3 5' - CTC CCT CTA TGT GTT TCT CC -3' 5' - GAA AAT CTT TCT ACC CTT CCA G- 3' 5' - GGA AGA AAA ATT GGG CCT TA - 3' 5' - CTC TCT ATC TCT GCC CCA - 3' 5' - TTC CCC ATG AAT GGA AGA AG -3' 5' - ATT GAT TCG ATG TGA GCA TCG - 3' 5' - GTT CGC TAG TTT GTT TGA TCC C- 3' 5' - TCC CAG CAA ATC CTT GAC TC - 3'
Vendramin etal., 1996
Vendramin etal., 1996
Vendramin etal., 1996
Vendramin etal, 1996
Vendramin etal., 1996
Vendramin etal., 1996
Echtefa/., 1999
Echi etal, 1999
Soranzoefa/., 1998
Soranzoefa/., 1998
KET QUA vA THAO LUAN
Tdng sd 10 cap mdi SSR da san sinh 21 allele tir 40 cay Thuy tiing tu nhien (Glyptostrobus pensilis) thu thap d 2 dia diem nghien cifu EaRal (EaH'Leo) va Trap K'So (Krdng Nang), trung binh 2,1 allele cho mdt locus SSR (A). Ti le phan tram locus da hinh (P) chiem 80%, vdi gia tri PIC dao ddng tir 0,008 (pt 110048) ddn 0,229 (pt 15169), trung binh 0,122. Tuy nhien, ti le phan doan giua cac cap mdi khac nhau, dao ddng tir 50% d ca 3 cap mdi pt 110048, pt87268 va P5 ddn 100% d 2 cap mdi ptl5169 va PI, trung binh 70,83%. Hai locus don
hinh dugc tim thay d pt26081 va pt30204. Gia tri PIC thap (<0,6) da chi ra mire do khac nhau ve di truyen giu'a cac ca the nghien ciru thap. Dieu nay cd the lien quan den lich sir sdng ciia loai. Thuy tiing la cay gd lau nam, sinh san don tinh va thu phan nhd gid. Ket qua thu dugc phii hgp vdi mgt sd cdng trinh nghien ciru ve di truyen quan the thdng da dugc cdng bd, nhu Piniis longaeva: A = 2,11 va P = 12,2%
(Hichert, Hamrick, 1983), Picea marlana: A = 2,2 va P = 69,2% (Wu et al, 1999). Tuy nhien, cac gia tri thap ciing dugc tim thay d mdt sd loai thdng Picea breweriana: A = 1,48 va P = 44,2%) (Ledig et al. 2005), Abies balsamea A = 1,21 va P = 20,5%
(Shea, Fumier, 2002) va cao d Pimis flexilis A = 3,7 va P = 95,2% (Jorgensen et al, 2002).
Tren ca sd phan tich he sd tuong ddng di truyen theo Jaccard, mdi quan he giila cac ca the nghien ciru dugc the hien tren so do hinh cay va bieu do tga do (Hinh 1) chi ra 3 nhdm ro rang. He sd tuong ddng di truyen dao ddng tir 0,643 den 1,0, trung binh 0,829.
Nhdm I gdm cac ca the thugc khu bao tdn thien nhien Trap K'So: EKI den EK17 cd mire do tuong ddng di truy6n dao dgng tir 0,643 (EK02-10) ddn 0,960 (EKOl-07), trung binh 0,768. He so tuong ddng di
truyen cao, tren 90%o d nhdm nay dugc tim thay d 3 cap ca thd EKOI vdi EK03, 07 va 12. Cac ca thd d khu bao tdn thien nhien EaRal dugc tach thanh 2 nhdm rieng biet (nhdm II va III). Nhdm II gdm 20 ca the vdi mire do he sd tuong ddng di truyen dao ddng til' 0,742 ddn 1,0, trung binh 0,775. Nhdm nay ti le he sd tuong ddng di truyen cao tren 0,90 chiem 30,7%.
Ba ca the EHOI-03 hinh thanh nhdm III vdi he sd tuong ddng di truyen cao nhat, dao ddng tir 0,917 den 1,0 trung binh 0,945. Cac ca the thudc nhdm nay deu cd he sd tuong ddng -vugt 90%).
Bang 3. Gia tri PIC va ti le phan doan da hinh ciia 40 mlu Thuy timg.
Cap moi SSR PIC Phan doan da hinh Phan doan dong hinh Ty le phan doan da \\\n\\
1 2 3 4 5 6 7 8 9 10 Tong
Pt 71936 Pt110048 Pt 87268 Pt 26081 Pt15169 Pt 30204 PI P3 P4 P5
0,109 0,008 0,204
-
0,229
-
0,124 0,178 0,109 0,013
4 1 1 0 3 0 3 4 4 1 21
2 1 1 1 0 1 0 1 2 1 10
66.67 50.00 50,00
100.00
100.00 83.33 66.67 50.00 56.67
II III
III
Hinh 1. Bieu dd hinh cay (A) va bleu dd toa do (B) cua 40 miu Thiiy tung tinh theo he s6 di truyin ciia Jaccard va kieu phan nhom UPGMA. EK: Trip K'So, EH: EaRal.
334
Tgp chi Cong nghe Sinh hoc 8(3): 331 -336, 2010
KET L U A N V A KIEN NGHI
Mire do da dang di truyen cia loai Thiiy tiing (Glyptostrobus pensilis) song tai khu bao tdn thien nhien EaRal (EaH'Leo) va Trip K'So (Krdng Nang) khdng cao, c6 the lien quan den lich sir sdng ciia loai va thu phan nhd gid. Mdt sd ca the cd quan he gan giii nhau ve mat di truyen deu xuat hien d ca 3 nhdm, dac biet cac ca the thugc nhdm III (he sd tuong ddng di truyen >90%). Dudi ap luc tang dan sd va yeu cau phat tren kinh te nhanh, rirng, noi sdng ciia loai dang bi de doa. Rirng bj pha buy de md rgng dat canh tac trdng cay cdng nghiep (chu yeu cay ca phe). Dat rirng bj suy giam manh va tao dieu kien thuan Igi cho cac loai cay bui va cd phat trien. Mdt vai quan the tu nhien ciia loai nay dang tdn tai trong nhirng manh riing bi phan cat va ddc lap vdi nhau. Sd lugng cay Thuy timg hien cdn rat it, khoang 300 ca the.
Hon niJa, do viec xay dap giir nude phuc vu cho cdng viec tudi tieu phuc vu cay ca phe vao miia khd, nude trong rirng ludn ludn d mire cao va tdn tai trong nhieu thap ky. Hien nay, cay cd dudng kinh dudi 10 m khdng dugc tim thay d ca 2 khu rirng dac dung Trap K'So (huyen Krdng Nang) va EaRal (huyen EaH'Leo). Chat cay Thuy tiing bdi mdt sd ngudi dan dia phuong van dang dien ra ca 2 khu Bao tdn, dac biet d EaRal. Dieu nay han che rat nhieu trong cdng tac bao tdn va phuc hdi loai. De bao tdn va phuc hdi loai cay Thuy tiing, chung tdi cd mdt sd kien nghi sau. Trudc tien, phai duy tri tinh da dang di truyen tai 2 khu Bao tdn thien nhien EaRal va Trap K'So, ngan chan chat, khai thac loai cay nay bang mgi hinh thirc. Thay ddi che do giii' nude trong riing de dan dan phuc hdi noi sdng ciia chiing, dac biet tao dieu kien cho cay con tai sinh. Cudi ciing, khdi phuc noi sdng cua chiing va dua cac cay con vao trdng. Muc dich nay se tao mdt quan the Idn hon dam bao duy tri tinh da dang di truyen cao va tiem nang tien hoa cua loai.
Loi cam on: Cong trinh duac hodn thdnh bai kinh phi ciia de dn BVMT ciia Bo Tdi nguyen vd Moi trudng: "Bdo ton vd sir dimg ben vimg mot so loai thong quy hiem co gid tri kinh te cao dang bi de doa tuyet chiing vd khu he ndm noi ky sinh co ich trong cdc lodi nghien cim ".
TAI LIEU THAM KHAO
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EVALUATION OF GENETIC VARIATIONS WITHIN GLYPTOSTROBUS PENSILIS POPULATIONS IN DAK L \ K USING SSR MARKERS
Vu Dinh Duy', Bui Thi Tuyet Xuan^, Tran Vinh^, Nguyen Minh Tam''' Vietnam National Museum of Nature
'Institute of Ecology and Biological Resources
The Western Highlands Agro-Forestry Scientific and Technical Institute
SUMMARY
Genetic variation and relationship of 40 Glyptostrobus pensilis samples collected from two locations. Trap K'Sor (Krong Nang) and EaRal (EaH'Leo) belonging to Dak Lak province, based on SSR markers. 10 of 21 tested SSR primers were used in this study. A total of 21 alleles were determinated, with a mean alleles per locus 2.1. PIC value (The Polymorphic Information Content) varied from 0,008 (ptl 10048) to 0,229 (ptl5169), an average of 0.122. Cluster analysis using unweighted pair group method with arithmetic average (UPGMA) revealed 3 groups from 40 the collected individuals in EaRal and Trap K'Sor. All of inviduals on Trap K'Sor clustered together with an average of genetic similarity coefficient of 0.768. Three individuals of EaRal was formed a group with the high genetic similary coefficient (>0.9). The implication of the results from the study to maintain genetic resources of G. pensilis has been discussed
Keyworks: conifers, conservation, genetic variation. Glyptostrobus pensilis, SSR
' Author for correspondence: Tel: (84)914539336; E-mail; m^lamfcihn.vnn.vn 336
