PHAIM T i C H D A DAIMG D l TRUYEIM L O A I CAIMG LO {BETULA ALIMOIOES DUCH-HAIVI) DAIMG C H I T H |
PHAIM TIJ R A P D V A c p S S R
Nguyin Hoing Nghla\ T r I n Thanh Trang2,Le Van Son^
Chu Hoang Hl^, Dd Tien Phlt^, Nguydn Van Phuong^
TOM TAT
Cang Id la loai cay co tiem nang ldn cho trdng rimg. Hai xuat xir ban dia va 3 xuat xir thu tii Trung Qudc la ngudn gien tdt cho phat trien loai cay nay. Phan tich ADN cua 130 mau cay Cang Id lay tii tap doan mau thu thap bing chi thi RAPD vdi 5 mdi nglu nhien cho thay ca 5 mdi deu cho tinb da binb ve phan doan ADN dupe nhan ban. Mirc dp da hinh PIC dao ddng tir 0,2223 - 0,6862. So dd tong the cua 45 gia dinh cho thay, cac gia dinh Cang Id cd xuat xir khac nhau cd mdi quan he di truyen rat phuc tap va cd sy phan biet ro rang giua cac vimg xuat xir. Cac gia dinh cua viing xuat xii Ceheng tuong ddi dn dinh va tap trung chu yeu d nhanh phu sd 4. Tuong ty nhu vay cac gia dinh cua xuat xir Chieng Bdm dn dinh va tap trung vao nhdm phu sd 8. Sd lupng mau tap trung nhieu nhat b nhanh phu sd 7, bao gdm cac gia dinh cua xuat xir Lingyun, Menghai va Cd Ma. Cac miu cua 3 vimg xuat xu: nay cd bien ddng ldn va nlm rai rac trong cac nhanh ciia cay phan loai. Cap mdi dac hieu cpSSR dung trong phan tich ADN luc lap da khdng chi ra dupe tinh da hinh ca khi phan tich bing cac enzym han che. Ket qua nhan dupe cho phep khIng dinh sy bao thu rat cao ve mat di truyen trong he gien luc lap cua Cang Id.
Tir khoa: Cang Id, da dang di truyen, AND da hinh phdng dai ngau nhien RAPD, cpSSR.
LDATVANDE
CIng Id (Betula alnoides Buch-Ham) II loli cay cd phan bd rdng fir Trung Qudc, Mien Dien, Lao, An Do, Thli Lan den Viet Nam (Zeng Jie etal, 1999). Cay gd nha, cd the cao 25 m, dudng kinh 60 cm. LI cay mgc nhanh, tien phong ua sing, ryng la. Tai sinh ty nhien tdt noi dlt trdng, chiu duge noi dlt khd, ngheo xlu. Thudng tham gia vio ning phuc hdi, tap tmng fhlnh d c quin the uu the. Gd cd fhlnh phln co ly tdt, dung llm vin lang vl ddng dd dung trong nhl. Trong nhung nam gin day, chi thi phan fir dugc sir dyng mdt cich rpng rai vl phd bien trong nghien cun ngudn gdc phlt sinh loli, phan loai, tim mdi quan he di truydn giffa cic loli, dinh gil mdi quan he di truyen trong loli v l xult xii. Bii viet trinh bIy ket qua phan tich da dang di truydn bing ky thuat chi thi phan hi RAPD v l cpDNA ddi vdi loli cay Cang Id vdi d c ngudn gidng dugc thu tai Viet Nam v l Trung Qudc.
IL VAT UEU VA PHUDIMG PHAP NGHIEN CUU 1. Vat lieu
Vat lieu phan tich he gien b i n g ky thuat RAPD:
130 miu CIng Id dugc su dung llm vat lieu nghien
' PGS. TS. Vien Khoa hpc Lam nghiep Viet Nam,
" Vien Khoa hpc Lam nghiep Viet Nam.
Vien Cong nghe Sinh hpc.
cim. CIc mau cd ngudn gdc fir 5 vung dia ly khac nhau (5 xult xir II Ceheng, Lingyun v l Menghai cua Trung Qudc, Cd Ma v l Chieng Bdm ciia Viet Nam) duge thu thap v l cho vio tui ni long mieng kin cung vdi silicagel hut am v l bao quin trong hi sau d -85°C hoac sir dyng ngay de tich chiet ADN.
Hoi chat vi thiet bi miy moc: Tag ADN polymeraza (Perkin-Elmer); mIy do quang phd Diod Array Spectrophotometer (My), mIy ly tam AvantiT^'30, mIy PCR-Thermal Cycler PTC 100 vl d c h o i chat sinh hgc phan fir khIc.
Cic mdi su dung trong phin img RAPD: Bao gdm 5 mdi nglu nhien su dung cho phan tich ADN bd gien cda 130 miu cay CIng Id do Phdng Cdng nghe Te bio Thyc vat - Vien Cdng nghe Sinh hgc cung d p .
Vat lieu phan tich he gien luc lap: 20 miu CIng Id dugc sir dung llm v | t lieu nghien cim. Mdt cap mdi dac hieu ddi vdi he gien lyc lap triiQ - triii (bang 2) duge mua tir hang IDT (cua My) de phan tich he gien lyc lap. CIc enzym han che BamFK, Haelll, HinA, Pstl va Taql duge dung de cat sin p h l m cua cap mdi luc lap.
2. Phuong p h i p
Tach chiet vi chuan do ADN: ADN cua 130 miu II CIng Id dugc tich chief v l tinh sach theo phuang p h i p cua Doyle v l Doyle (1990) cd cli tien. ADN sau
khi tich chiet dugc kiem tra tren gel Agaroza 0,8%, xlc dinh h i m lugng tren mIy do quang phd vl pha loang ve ndng do su dung 10 ng/pl.
Phan ung PCR-RAPD. Mdt phln ung PCR cd the tich 25 pi bao gdm: IX dich dem PCR; 2,5 niM MgCl,; 2 mM dNTPs; 200 nM doan mdi RAPD; 0,125 don vi Tag polymeraza vl 10 ng ADN khuon. Phan ung PCR- RAPD thyc hien trong mIy PCR- Thermal Cycler PTC 100 theo chu ky nhiet: Budc 1: 94"C - 3 phut; budc 2;
92''C - 1 phut; budc 3: 35»C - 1 phut; budc 4: 720C - 1 phut; budc 2 den budc 4 dugc lap lai 45 chu ky; budc 5:
72"C - 10 phut; budc 6: giu' d 4"C (Ravinsanka va cs, 2000; Tnrong Quang Vinh va cs, 2008).
Phan ung PCR- SSR: Mot phln ung PCR-SSR cd tdng the tich II 25 pi chira: IX dung dich dem PCR, 50 mM MgCla, 2 mM dNTPs, 10 ng mdi lyc lap, 0,5 don vi Taq polymeraza vl 50 ng ADN cua bg gien.
Chu trinh nhiet cua cua phan ung bao gdm d c budc:
budc 1: 94»C - 2 phut; budc 2: 94'^C - 1 phut, budc 3:
54"C - 1 phut; budc 4: 72°C - 1 phut; budc 2 den budc 4 dugc lap lai 34 chu ky; budc 5: 72''C - 10 phut; budc 6: giu san pham d 4''C (Heinze, 2007).
Phan ung cdt enzym han che: Sin pham PCR- cpSSR dugc cit bang d c enzym ban che vdi fhlnh phan nhu sau: 10 pi sin pham PCR, 2 pi lOX buffer, 2 yl enzym vl 6 yl dd H2O. Phan ung d t duge thyc hien d 37°C trong thdi gian 16 gid.
Phin tich so lieu: Theo quy udc: 1= phan doan ADN xult hien vl 0 = phan doan ADN khdng xult hien, khi dien di sin pham RAPD vdi d c mdi nglu nhien. Xac dinh he sd tuong ddng di truyen theo phuong phap cua Nei va Li (1979).
Sij =
2a 2a-\-h-\-c
Trong dd Sij he sd gidng nhau giua hai c l the / vly; a: Sd phan doan ADN xult hien d ca d the ivaj, b: Sd phan doan ADN xuat hien d / v l khdng xuat hien bj c: Sd phan doan ADN xult hien dy vl khdng xuat hien b i
Lap bieu dd hinh cay trong chuong trinh NTSYSpc 2.0 Games, 1998). Him lugng fhdng tin tinh da hinh (Polymorphism Information Content = PIC) (Marilyn and Jose, 2002) cua mdi mdi xlc dinh theo cdng thirc PIC = 1- Z P / , trong dd P/ la tan sd ciia alen thu / cua kieu gien dugc kiem tra. Pham vi gia tri PIC tu 0 (khdng da hinh) tdi 1 (da hinh hoan
toln). Thi nghiem dugc thyc hien tai Phdng Cdng nghe Te bio Thyc vat - Vien Cdng nghe Sinh hgc.
III. KET QUA NGHIEN CUU
1. Ket q u i phan tich he gien bang chi thi RAPD Sd phan doan vi tan sd xuit hien cac phin doan:
Sin pham RAPD vdi d c mdi khIc nhau dugc dien di tren gel agaroza 1,8% de phan tich tinh da hinh ADN cua 130 mau cay nghien cim. Kich thudc d c phan doan ADN dugc nhan b i n trong khoing hi 250-2000 bp. Tinh da hinh the hien d sy xult hien hay khdng xuat hien cua d c phan doan khi so sinh giua cac miu vdi nhau trong cung 1 mdi. Ket qua tdng hgp dugc the hien tren b i n g 3. Khi dung 5 mdi cho phan tich cac mau Cang Id, ket qua cho fhly c l 5 mau deu cho tinh da hinh ve d c phan doan ADN dugc nhan bin. Ty le phln tram phan doan da hinh cua d c mdi dao ddng tu 28,57-88,89%, trong dd cao nhat II mdi RA159 va thap nhat II mdi OPH08. Dieu nly cung phu hgp khi phan tich gil tri PIC ve muc dp da hinh giua cac mdi, gil tri PIC cang cao mirc dp da hinh cua mdi d n g ldn. Ket qua d b i n g 1 cho fhly, gil tri PIC dao ddng tii 0,2223-0,6862, cao nhlt II mdi RA159 va fhlp nhat II mdi OPH04. Gil tn PIC khdng chi phln Inh ty le d c phan doan da hinh ma cdn dinh gil muc do khac biet ve phan doan nhan dugc giua cac miu. Trong 5 mdi cho tinh da hinh cd hai mdi RA46 vl RA159 cho gil tri PIC > 0,5 the hien tinh da hinh cao cua mdi sir dung.
Bing 1. Tinh da hinh ve phan doan ADN dugc nhan ban cua 5 mdi phan tich
Mdi OPH04 0PP19 RA46 RA159 OPH08
% phan doan da hinh 33,33 37,50 66,67 88,89 28,57
PIC 0,2223 0,4759 0,5634 0,6862 0,3215 Cac sd lieu sd phan tich PCR-RAPD dugc xir ly va phan tich trong chucmg trinh NTSYSpc version 2.0 nhlm tim ra khoing d c h di truyen giua d c miu cay nghien cim fhdng qua he sd tuong ddng di truyen vl bieu dd hinh cay. De kiem tra phuang phip phan nhdm, gil tri tuong quan kieu hinh theo ba phuong phip tinh he sd di truyen gidng nhau (phuong phip cua Jaccard, cua Nei & Li, cua Sokal) vdi bdn kieu phan nhdm (WPGMA, UPGMA, lien kef holn toln vl lien ket dem le) (bang 2) da duge xlc dinh. Bieu dd hinh cay dugc thief lap dya tren gil tri tuong quan cao nhat vdi d c gil tri khi r > 0,9: tuong quan rat NONG NGHIEP VA PHAT TRIEN NONG THON - SO 2 - THANG 2/2010 109
chat, r = 0,8 - 0,9: tirong quan chat, r = 0,7 - 0,8: tuong quan tuong ddi chat, r < 0,7: tuong quan khdng chat.
Bing 2. Gil tri tirong quan kieu hinh (r)
SM Dice Jaccard
UPGMA 0,92029 0,92328 0,95507
WPGMA 0,92029 0,93328 0,94576
Lien ket hoan toan
0,91632 0,93035 0,93282
Lien ket don le 0,91571 0,89814 0,88383 Kef qui bing 2 cho thay, vdi ba d c h tinh he sd di truydn gidng nhau vl bdn kieu phan nhdm phan anh mdi tuong quan kieu hinh cua 130 miu Cang Id dao ddng fu fu'ong quan chat (0,88383) tdi rat chat (0,95507). Trong dd gia tri tuong quan kieu hinh (r) ldn nhlt 0,95507 khi tinh fheo he sd di truyen Jaccard va kieu phan nhdm UPGMA. Vi vay, sa dd hinh cay (Hinh 1) dugc thief lap theo he sd di truyen gidng nhau Jaccard va kieu phan nhdm UPGMA.
Hinh 1. Cay phan loai chi tiet ve mdi quan he di truyen cua 130 m i u CIng Id
Qua bieu dd hinh cay (Hinh 1) cho thay, phln ldn cac miu thudc d c gia dinh khIc nhau cd muc dd tuong ddng cao. Ket qua da chi ra 5 gia dinh khdng
cd su sai khac giua cac c l the khi phan tich vdi 5 mdi nglu nhien dd la gia dinh 26/16, 26/23, 15/39, 27/16 va gia dinh 27/20. Ngoai ra d c cap mau (15/21-1 va 15/21-3), (27/13-3 va 27/23-1) va (CBll-3 va CBll-2) cung khdng cd sy sai khIc ve sd phan doan ADN dugc nhan ban.
Bing 3. Danh sleh 45 miu CIng Id dung trong phan tich tdng the (CB: Chieng Bdm, CM: Cd Ma, 26:
Ceheng, 27: Menghai, 15: Lingyun) TT
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Ky hieu CB3-1 CB8-2 CB92 CBll-1 CB12-1 CM9-2 CMll-3 CM 12-2 CM13-1 CM17-1 CM 18-2 CM21-3 CM22-3 CM25-3 26/2-3
TT 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Ky hieu 26/13-3 26/14-3 26/15-1 26/16-1 26/18-2 26/19-3 26/20-1 26/23-3 26/25-2 15/20-2 15/21-3 15/22-2 15/23-1 15/25-2 15/28-1
TT 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Ky hieu 15/31-2 15/33-2 15/36-2 15/38-3 15/39-2 15/49-1 27/5-1 27/11-1 27/13-3 27/15-3 27/16-1 27/20-1 27/22-2 27/28-3 27/30-2 Tuy nhien, viec quan sat cay chi tiet cua 130 mau rat khd de xlc dinh mdi quan he cua d c gia dinh d d c khu VU'C khIc nhau. Do vay can cir vio so dd chi tiet vdi 130 miu, 45 mau (Ba; -g 5) dai dien cho 45 gia dinh Cang Id vdi 5 vung xuat xu khIc nhau da dupe lya chpn de xay dtmg cay phan loai tdng the (Hinh 2).
So dd tdng the (hinh 2) cho thay, d c gia dinh CIng Id cd xuat xir khIc nhau cd mdi quan he di truyen khac biet nhau. Cu the cay phan loai chia llm hai nhlnh chinh: n h l n h 1 chi cd gia dinh sd 15/23
(xuat xu Lingyun) vdi muc do sai khIc so vdi cic mau cdn lai la 45% (1-0,55), nhanh hai gdm 44 mau cdn lai, mtic do sai khIc dao ddng tu 5% (1-0,95) den 44% (1-0,56). Hai miu cd dd tuong ddng cao nhlt II 15/36 v l 15/38 vdi muc do tuong ddng II 95% cho thay cd the chung cd cung ngudn gdc cay me.
Vdi 5 mdi RAPD budc diu cung da chi ra dupe sy khac biet k h i ro rang ve quan he di truyen giiia d c gia dinh Cang Id cd xult xu khac nhau. CIc gia dinh cua vung xuat xu Ceheng tuong ddi dn dinh va tap trung chu yeu d nhanh phu sd 4. Tuong tu nhu v | y d c gia dinh xuat xu Chieng Bdm dn dinh vl tap trung vio nhanh phy sd 8. Sd lugng miu tap trung nhieu nhat d nhanh phu sd 7 bao gdm cac gia dinh
cua xuat xu Lingyun, Menghai va Cd Ma. Cac mau khac trong vung xult xu Lingyun, Menghai vl Cd Ma cd bien ddng ldn va n l m rli rac trong d c nhlnh cua cav ohan loai.
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-«C,JJ -r.mn -s.o-i
4:
—1;/3?-3 iM.l itm.l li/31-3
— u a i - 1 l-'IS-l 1!/51,1 LSS'-l iM|,|
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—ufn.i
-KJ13,3 -»1J-1 -»J1«,1
-m-i -cras,i
-CMIM
Hinh 2. Cay phan loai tdng the ve mdi quan he di truyen cua 45 gia dinh CIng Id
2. Ket q u i phan tich he gien luc lap bang cpDNA Cap mdi lyc lap trnD-trnT da dugc, dung de phan tich sy da dang di truyen cua 20 mau CIng Id d muc dp he gien lyc lap. Trong qua trinh chay PCR, nhiet dp bit mdi tdi un cho cap mdi nay ddi vdi CIng Id II 52°C. San pham PCR cua cap mdi trnD-trnT vdi dd dai Idiolng 1600 bp. San pham cua cap mdi nay deu cd dp dli tuong fy nhau d fat d 20 mau Cang Id nghien cun. Nhu vay, ve kich thude sin pham PCR cua mdi luc lap khdng cho thay sy da hinh giua cac miu nghien cim.
Mdt sd enzym ban che BamHI, Haelll, HinA, PsH vl Taql da dugc dung cat d c san pham nhan dupe sau khi nhan bin d c vung cpDNA bing cap mdi tuong ung. Neu d d c miu cd sy khIc nhau vd trinh ty nucleotit thi se din den thay ddi vi fri cat cua cic enzym. Bing d c h nly cd the nhan dugc sy da hinh trong cpDNA d d c mau nghien ciru. Tuy nhien ddi vdi loli CIng Id, sin pham PCR cua cap mdi nghien cun dugc d t bdi cac enzym ban chd d tren khdng cho da hinh d tat c l cac mau. Cu the II cd enzym thi d t dugc nhung khdng cho da hinh
{BamHI, Taql), cd loai thi khdng d t dugc (Haelll, HinA). Vi vay vcii chi mot cap mdi trnD-trnT thi chua the nghien cun tinh da dang di truyen d muc dp he gien luc lap cua loai. Do dd de cd dugc hue tranh cy
the va day du hem can phai khao sat vdi nhieu cap mdi lyc lap hon vl sd lugng enzym han che cung cin dugc tang len. Tuy nhien, vdi ket q u i nly cd the di den mdt ket luan sa bd II cac mau cua loli nghien ctru la rat bao thu d locus trnD- trnT. Ndi khac di II d vi tri nly giu'a d c mau cua loai cd dp tuong ddng vd mat di truyen d he gien lyc lap 11 rat cao. Hon nua, fheo ban chat cua he gien luc lap fhi day la he gien rat it cd nhimg bien ddng giua d c c l the cung loli.
IV. KET LUAN
1. Phan tich ADN cua 130 mau cay CIng Id lay tir tap doan mau thu thap bing chi thi RAPD vdi 5 mdi ngau nhien cho thay d 5 mdi deu cho tinh da hinh ve phan doan ADN dugc nhan ban. Mirc dd da hinh PIC dao ddng tir 0,2223-0,6862. Budc diu da xay dyng dugc cay phan loai cua 130 miu cay Cang Id nghien cun, trong dd cd 5 gia dinh khdng cd sy sai khac giua d c c l the klii phan tich vdi 5 mdi nglu nhien dd II gia dinh 26/16 vl 26/23 (xult xu Ceheng), 15/39
(Lingyun), 27/16 vl 27/20 (Menghai).
2. So dd tdng the cho 45 gia dinh cho fhly, d c gia dinh Cang Id cd xuat xu khIc nhau cd mdi quan he di truyen rlt phuc tap v l cd sy phan biet rd ring giua d c vung xuat xir. CIc gia dinh cua vung xuat xii Ceheng tuong ddi dn dinh v l tap trung chu yeu d nhlnh phy sd 4. Tuong tu nhu vay d c gia dinh cua xult xu Chieng Bdm dn dinh va tap trung vio nhdm phu sd 8. Sd lugng mau tap trung nhieu nhlt d nhlnh phy sd 7 bao gdm d c gia dinh vdi xuat xir Lingyun, Menghai v l Cd Ma. CIc miu trong vung xult xu Lingyun, Menghai vl Cd Ma cd bien ddng ldn va nlm rli rac trong d c nhlnh cua cay phan loai.
3. Cap mdi dac hieu cpSSR dung trong phan tich ADN luc lap trnD-trnT nhan dugc doan ADN mong mudn d nhiet do bit mdi la 52''C ddi vdi CIng Id. CIc enzym Taql va BamHI d t dugc d c sin pham cua cle cap mdi trnD-trnT b d c mau CIng Id nhung khdng cho da hinh, d c enzym cdn lai khdng d t dugc d c miu nghien ciiu. Ket q u i nhan dugc cho phep khIng dinh sy bao thu rlt cao ve mat di truyen trong he gien lyc lap cua CIng Id.
TAI UEU THAM KHAO
1. Doyle J. J. and Doyle J. L. (1990). Isolation of plant DNA from fresh tissue. Focus 12: 13-15.
2. Heinze B. (2007). A database of PCR primers for the chloroplast genomes of higher plants. Plants Methocls2007:3-4. •
NONG NGHIEP VA PHAT TRIEN NONG THON - SO 2 - THANG 2/2010 111
3. James R. F. (1998). NTSYSpc Numerical Taxonomy and Multivariate Analysis System Version
2.0. Exeter software Setiiuket, New York: 11733-2870.
4. Marilyn W. and Jose C. (2002). Data analysis in the CIMMYT Applied Biotechnology Center for fingerprinting and Genetic.
5. Nei M., W. H. Li, (1979). Mathematical model for studying genetic variation in terms of restriction end nucleases. Proc Natl Acad. Sci USA 76. 5269- 5273.
6. Nguydn Due Thanh, Nguydn Thuy Hanh, Nguydn Hoing NghIa (2005). Nghien cun quan he di truyen cua mdt sd loli thudc hp Dau (Dipteroearpaeeae) d Viet Nam dya tren da hinh ADN cua bd gien va luc lap. Ky yeu Hgi nghi toan qudc "Nhung van dd nghien cun ca ban trong khoa hgc sy sdng". Nhl xult ban Khoa hgc vl Ky thuat, HI Ndi, 2005. 1379-1382.
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thi phan tii RAPD. Tap chi Ndng nghiep vl PTNT, 14/2007, 44-48.
8. Nguydn Hoang NghIa, Tran Van Tien, Nguydn Thuy Hanh, Nguyen Due Thanh (2006). Ket qui phan tich da dang di tmyen loli Sao hinh II tim (Hopea cordata Vidal) thudc ho diu (Dipferocai-paceae) bang chi thi phan tir. Thdng tin Khoa hpc Ky thuat Lam nghiep, 1:1-6.
9. Ravinsanka K., V., Lahitha A., Dinesh M.R.
(2000). Assement of genetic relatedness among mango culfivals of India using RAPD marker. Hort Sci Biot: 87 - 89.
10. Tnrong Quang Vinh, Nguydn Thi Tam, Dd Tien Phlt, Nguydn Thanh Danh. (2008). Dinh gia sy da hinh ADN mdt sd gidng khoai tay (Solanum tuberosum L.) b i n g ky thuat RAPD. Tap chi Ndng nghiep vi PTNT, sd 1: 20-25.
11. Zeng Jie, Zheng Haishui vl Weng Qijie, 1999.
Betula alnoides - A Valuable Tree for Tropical and Warm-Subtropical Areas. Forest, Farm and Community Tree Research Reports-Vol. 4 (1999).
GENETIC DIVERSTFY ANALYSIS OF BETULA ALNOIDESBUCR-HAM. BY RAPD AND cpSSR MARKERS
Nguyen Hoang Nghia, Tran Thanh Trang, Le Van Son, Chu Hoang Ha, Do Tien Phat, Nguyen Van Phuong
Summary
One hundred thirty (130) leaf samples collected from Betula alnoides Buch-Ham provenance trial established with different provenances (three from China and two from Vietnam) were genettcally analysed by molecule markers (RAPD and cpSSR) in order to suggest suitable measures for genetic consei'vation of the species in the future. All 5 RAPD markers gave polymorphic DNA bands. PIC values ranged from 0.2223 to 0,6862. Separate analysis of 45 families have shown that families of different provenances have very complex genetic relation but there is a clear difference between provenances. Families of Ceheng provenance are quite stable and concentrated on branch No. 4. Similarly families of Chieng Bom provenance are stable and concentrated on branch No. 8. Samples concentrated on branch No. 7 come from families of Lingyun, Menghai and Co Ma provenances. Families of these provenances show high variability and occupied places on different branches. The cpSSR marker used in molecular analysis of ten representative samples did not give polymorphic DNA bands, even when using restricted enzymes. This means that the genetic content in chloroplast DNA of Betula alnoides Buch-Ham is very highly conservative.
Keywords: Betula alnoides Buch-Ham, genetic diversity, cpSSR, Random amplified polymoiphic DNA RAPD.
Ngudi phln bien: TS. La T u l n Nghia
