NAM DT/A

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HOl NGHI KHOA HOC T O A N Q U O C Vfe SINH THAI V A TAI NGUYEN SINH VAT LAN THCf BA

KHAC BIET DI TRUYEN CUA ONG APIS CERANA TRONG VUNG BAO T 6 N ONG VIET NAM DT/A VAO DA HINH TRINH TV GEN COI CUA TY THE

LE QUANG TRUNG, NGUYEN DlTC LAM, DINH THI DAN NGUYEN NGQC V U N G , PHUNG HlTU CHINH Trung tdm Nghien ciru vd Phat trien Ong

NGUYEN T U 6 N G VAN, VU THI HIEN Vien Cong nghi sinh hpc Cimg vdi bio tdn ngudn gen nhieu loii dgng vat khac, bio ton ong mat da dugc nhi nude ta chii trgng dau tu tir cudi nhimg nim 80 cua the ky 20. Trong sd 5 loii ong mat bin dja thugc doi tugng bio tdn gdm Apis lab'oriosa, A.dorsata, A. cerana, A.florea v i A. andreniformis, chi co A. cerana cd the thuan dudng v i nuoi dugc. Vi vay loii ong niy khdng chi dugc bio ton de duy tri da dang sinh hgc vi cin bang sinh thii nhu 4 loii khic m i cdn de cung cap ngudn nguyen lifu khdi thuy eho cdng tic chgn tao giing phit trien nginh ong. Hifn nay, ong A. cerana dang dugc bao ton d 5 vimg: Tiia Chiia (Difn Bien), Dong Van (Hi Giang), Nguyen Binh (Cao Bing), Ban Sen (Van Ddn, Quing Ninh) va Cdn Dao (Bi Rja-Vung Tiu). Cimg vdi vifc hudng din ky thuat quan ly vi phit trien din ong de duy tri da dang di truyen v i tie dgng ehinh quyen dia phuang ngan cam du nhap ong vio vimg bio ton gay lai tap gidng, danh gii ngudn gen bio ton ludn dugc di kem. Trong do dinh gii khic bift ve di truyen cua cie quin the ong giira cic vimg bao ton v i da dang di truyin trong mgt viing bao ton dugc thye hien hing nim de khang djnh hifu qui bio tdn. Cho den nay nhifm vy niy ehii yeu dya vao cic ehi tieu hinh thai nhu chieu dii vdi, kich thudc gde cinh, guang sap... Tren thye t i , sir dung chi tieu hinh thii cho myc dich niy cd nhieu ban che do tinh da hinh thip, sai s i khi do dim cao, nen rit khd de phan bift dugc muc dg dudi loii. Ky thuat phan tu dya vio da hinh cua enzyme, vi du, mgt phan nao da khac phyc dugc nhiing yeu diem ciia phuang phap dya vao hinh thii. Tuy nhien, si enzyme cho da hinh khdng nhieu vi tmh da hinh cua enzyme cung khdng cao. Trong nhung nim gin diy, ky thuat phan tii dya vio tinh da hinh ciia ADN ty thi (mtDNA) da dugc img dyng rgng rai v i hieu qui de khac phuc nhung ban che ciia hai phuang phap tren.

Vi cic die tinh.cua gen tren mtDNA di truyin theo ddng mg, thudng khdng ed qui trinh tai td hgp v i mire dg dgt biin cua mtDNA cao ban so vdi ADN d nhan, nen phan tich da hinh ciia mtDNA la ^iii phip hifu qui v i chinh xic de xic djnh da dang di truyin trong quin thi vi khac biet di truyen giira cic quin thi. Tuy nhien, d cie viing khic nhau tren mtDNA tic dg dgt bien cung khic nhau. Cho nen vifc lya chgn gen hay viing tren mtDNA tuy thugc vio myc dich nghien cuu. Tir khi toin bg hf gen tren mtDNA cua Apis mellifera da dugc giii trinh ty, phan tich thanh phin v i xic djnh vj tri gen, nhiiu ky thuat phan tir dua vio tinh da hinh d cic viing, cic gen khic nhau tren mtDNA dugc irng dyng rgng rai d i nghien cuu quan hf chimg loai, quan he di truyin va qua frinh tien hoi ciia cac loai ong mat d mirc dg loii, trong loii vi quin thf.

Trinh tu cua doan tren gen COI, COU dugc ung dung d i nghien cuu v i khac bift di fruyin va da dang di truyen ciia ong A. mellifera a Chau Au.

Cl nude ta, da cd mgt so nghien ciru quan hf di truyen ciia ong A. cerana dya v i da hinh ciia mtDNA. Tir kit qua ting hgp nhieu nghien curu ve ong A. cerana dya vio phin tich hinh thai, da hinh cua men vi mtDNA cua Hepbum v i cgng sy cd thi nhan ra ring d nude ta cd thi tin tai 3 phan loai A. cerana, bao gdm A. c. cerana a dgc bien gidi Ddng Bic, A. c. indica a phia Bic, d phia Nam cd the li A. c. indica hoic li A.c.javana. Thii dya vao da hinh frinh tu mtDNA de

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HOI NGHI KHOA HOC TOAN QUOC Vg SINH THAI VA TAI NGUY£N SINH VAT LAN THCf BA nghien ciru da dang sinh hoc eiia/I. cerana a mien Bac v i mien Nam. Tuy nhien s i lugn^ mau qua it, khong dii dai dien de phan tich da dang di truyen trong quan the va doan dugc giai trinh tu lai qua ngan, khdng dii dot bien dac trung de phan tich khac biet di truyen giira cac quan thl.

Han nira, hai quan the lai dugc phan tich dya vio hai vj tri khic nhau tren mtDNA di phan tich (COI cho A. cerana phia Bac va doan giira COI-COII cho A. cerana phia Nam) nen khong thi so sinh dugc. Trong nghien ciru niy cua chiing tdi, da dang di fruyen trong mdi vimg bio tin ciing nhu khic bift di truyen giira 5 vimg bao ton ong A. cerana se dugc dinh gii bing each phan tich da hinh ve trinh ty ciia doan khoing lOOObp tren gen COI ciia mtDNA.

L PHUONG PHAP NGHIEN CUtJ

Mau ong thg dugc thu giira nam 2008 vi 2009 tir 39 din ong A. cerana (AC) dang nuoi giir d 5 viing bio ton trong c i nude (hinh 3), gdm 8 mau d Tiia Chiia (TC)- Dien Bien, 10 mau d Ban Sen (BS)- Quing Ninh, 6 mau d Cdn Dio (CD)- Bi Ria-Viing Tau, 7 mau d Dong Van (DV)- Ha Giang, 8 mau d Nguyen Binh (NB)- Cao Bang va mgt mau ong A. dorsata a Mgc Chau (MC AD) de lim nhom ngoai khi phan tich. Cir 10 din ong thu 1 mau tir mgt din bat ky.

Moi mau thu tir 5-10 ong thg, ngam vio cdn tuyft doi vi giir d ^''C cho den khi tich ADN.

Chgn ngiu nhien 1 ong thg/mau de tach ADN. Xir ly mau theo Gamery vi cs. vi tich ADN tong so tir phin co ngyc theo kit tach ADN ciia hang Fermentas (Du:c).

Thiet ki moi PCR sir dyng phan mim ADNMAN 4.0.1.1 (Lynnon Biosoft). Cap moi dugc thiet ke chung cho Apis cerana va A. dorsata, ed ma hieu va trinh ty: FTr_Acd: 5'- TTCCTAATTGGAGGTTTTGGAAATTG-3' vi RTr_Acd: 5'-ATAAATATTATAATA AATTGAATTTTTA-3'). Nhan bin doan gen dich, sir dung kit PCR cua hang Fermentas (Dire), Chu trinh PCR gim budc biin tinh ban diu 96°C trong 5 phut, tiip tyc 30 chu ky: 95°C - 0,5 phiit, 55°C - 1 phut, 72°C - 1,5 phiit; kit thuc chu ky cuii d 72°C trong 10 phiit. Nhugm sin pham PCR bang Ethidium Bromide, quan sat bang soi tia UV va so sanh kich thudc vdi thang ADN chuan (Invitrogen^'^, GeneRuler).

Sin phim PCR dugc gin vio vector pTZ57R/T (Fermentas, Due), nhan ddng trong vi khliin. Plasmid ADN dugc tach bing kit GeneJET^"^ Plasmid Miniprep Kit, cit bing EcoRJ vi Hindlll vi dugc giri sang Cdng ty Macrogen (60-24, Gasan-dong, Geumchun-gu, Seoul 153- IM, Korea) de giai trinh ty nucleotide 2 chiiu su dung cap mdi Ml3.

Phan tich mii quan hf vi da dang di truyin giira cic trmh ty tuang dong sir dyng phan mem Mega3.1 va ADNSp 4.10.9. Trong do, da dang di truyin trong moi quin thi dya vao da dang vi haplotype (Hd) va khoing eich di truyen giira cic trinh ty hoac nhom trinh ty (d). Trong khi khac bift di truyen giiJ-a cac quin thi (Gst) dya vao so sanh da dang haplotype trung binh giira cac quin thi vi khoang each di truyen (d) giii:a cac quin the. Khoing eich di truyen tinh theo mo hinh Kimura hai thdng si (Kimura 2-parameter distance). Gia fri Bootstrap dugc tinh tren co so 1000 lin nhic lai fren chuang trinh Seqboot. Sau do chuang trinh ADNDIST se ude lugng sy sai khic giira cac trinh ty dya vio nhirng dgt biin diim d ci 3 vi tri ma hoi vi khdng ma hoi cua moi bg ba ma hoi theo md hinh.

H. KET QUA NGHIEN ClTU

Da nhan bin thinh cdng doan ADN dich thugc gen COI ciia tit ci 40 mau ong nghien ciru, vdi kich thudc 1040bp. Dii chiiu, so sanh trinh ty nghien ciru ciia 39 mau A. cerana, da thong ke dugcl33 vi tri da hinh frong ting sd 1040bp (hinh 1). Sy bien dii tiin hda ciia cac vi tri da

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HOI NGHI KHOA HQC TOAN QUOC V£ SINH THAI VA TAI NGUYEN SINH VAT LAN THLf BA hinh se phan inh quan hf di fruyen giira cac quan the ong A. cerana cua cic vimg bio tin, ciing nhu da dang di truyen frong timg vimg bio tdn.

r , 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 1 4 4 5 6 6 8 9 9 9 9 0 1 3 5 5 6 6 7 7 7 7 8 9 0 1 1 2 3 3 3 4 7 1 2 3 3 3 4 6 6 7 7 8 2 5 5 6 7 7 7 8 0 0 2 4 5 5 7 9 9 9 0

3 4 5 3 8 1 0 6 7 0 4 6 9 5 4 8 6 9 2 6 1 4 7 8 3 8 1 0 1 8 1 2 7 6 6 2 1 0 1 3 8 0 3 5 8 8 3 0 9 8 1 4 7 3 4 7 8 6 2 8 3 4 5 7 0 CCTTATATATTGTTTTAAAGTTTTTACCAGCCTTAAACTTAAACATCAATATATTATITT

TC . =F * . . . . * . . * TC

TC TC TC TC TC TC BS BS BS BS BS BS BS BS BS BS CD CD CD CD CD CD DV DV DV DV DV DV DV NB NB NB NB NB NB NB HB

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T T . C .GCG.CA.A.

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HQI NGHI K H O A H Q C T O A N Q U O C V £ SINH T H A I V A TAI N G U Y g N SINH V A T L A N THCf BA 1 1 1 1 1 1

6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 0 0 0 0 0 0 0 1 2 4 5 6 9 9 9 0 0 1 2 5 6 6 7 9 0 2 3 7 7 8 8 9 0 0 1 1 2 4 4 6 7 7 7 9 9 9 9 0 1 1 2 2 3 9 5 1 5 1 9 0 3 9 2 8 4 9 9 5 8 1 6 7 2 7 0 6 2 5 7 0 9 2 5 5 5 8 3 5 8 8 0 1 3 9 5 1 2 0 6 8 TC

TC TC TC TC TC TC TC BS BS BS BS BS BS BS BS BS BS CD CD CD CD CD CD DV DV DV DV DV DV DV NB NB NB NB NB NB NB NB

C A A T T A A T T r A A T A T T T G T A A A C T T C T T T T T T A T T A T T T A T T G C T C r

* *

C . . .GTAA.G ..TAA.G ..TAA.G ..TAA.G . .TAA.G . .TAA.G .GTAA.G . .TAA.G .GTAA.G ..TAA.G T.T. . . . T.T T.T T.T T.T T . T . . * .

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Hinh I: 133 vj tri da hinh nucleotide khi so sanh cac doan COI dii 1040bp ciia 39 mau A.

cerana. Cac dau chiim theo hing dgc l i cic nucleotide giong voi trinh t y chuan ff hang thir nhat. Ky hifu * tren nucleotide cua trinh ty dau tien ciia moi viing bao ton l i vj tri

dft bien diem d i e trung cho viing bao ton

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HOI NGHI KHOA HQC T O A N Q U O C V ^ SINH THAI V A TAI N G U Y S N SINH VAT L^N THLf BA

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Hmh 2: Cay phat sinh chiing loai NJ (Neighbour Joining) the hifn khoang each di truyen giira 5 viing bao ton ong/I. cerana va da dang di truyen trong raoi viing ong bao ton. Cac so canh nhanh cac

cay chiing loai li gia trj bootstrap. Cac so cuoi moi nhanh li so thirty ciia mau

Ket qui phan tich quan hf di truyen bang phucmg phap NJ cho thay 39 trinh ty thugc gen COI cua ong A. cerana phan hda thinh 4 nhinh rieng bift tren cay phit sinh chung loai (hinh 2).

Dai difn A. dorsata nhu mgt nhdm gde, ed khoing cich di truyen tach bift so vdi ong A. cerana bao ton. Quan hf di truyen cua cic vimg dugc sap xip theo nhanh tir vimg Ddng Vin-Nguyen Binh den Ban Sen, Tiia Chiia vi Cdn Dao. Nhinh 1 gdm cac miu d viing bio tdn Nguyen Binh va Ddng Van, nhanh 2: Ban Sen, nhanh 3: Tiia chiia v i nhanh 4: Cdn Dao. Gii trj bootstrap giiJa cic nhinh khi cao tir 96-100%.

Phan tich khoing each di fruyen theo Tamura va Nei (bang 1) da chi tiit hem kit qui phan tich theo phuong phip NJ. Trong 5 vimg bao tin, khoang each di truyin eiia cie din ong A.

ceranO: a Cdn Dao li thap nhit (0,1%) va d Tiia Chiia li cao nhit (1,3%). Khi so sinh cap giira eic viing bao ton, khoang each di truyen giua eic cap vung dao dgng tir 0,24% din 5,83%. Gii tri khoang cich di truyen thip (D=0,024) eiia ong A. cerana giira hai viing Ding Vin va Nguyen Binh ehung td quan hf di truyen gan giii giira chiing.

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HQI NGHI KHOA HQC T O A N Q U O C V £ SINH T H A I V A T A I N G U Y £ N SINH VAT L A N THLf BA

Ket qua phan tich khoang each di truyen trong viing bao ton (d) va giira cac viing bao ton (D) theo Tamura va Nei. se, SE: sai so chuin

Bdng I

TC BS CD DV NB

Khoang each di truyen trong moi vimg bao ton (d±se)

1 0.013±0.002 0.002±0.001 O.OOliO.OOI D.002±0.001 0.003±0.001 Khoang each di truyen giCra cac viing bao ton (Z>±SE)

TC BS CD DV NB

0.0379±0.0057 0.0527±0.0066 0.0532±0.0070 0.0539±0.0070

0.0448±0.0062 0.0421±0.0064 0.0429±0.0064

0.0583±0.0071

0.0586±0.0071 0.0024±0.0007

Bdng 2 Ket qua phan tich khac bift di truyen giira cic ong viing bao ton A. cerana (Gst)

va da dang di truyen trong raoi vimg (Hd) theo Nei

TC BS CD DV NB

tt 5 viing bao ton N

8 10 6 7 8 39

h

1 1 2 4 5 25

H,

0.96 0.91 0.60 0.81 0.78 0.95

C„

TC BS

0.0325*

CD 0.117**

0.129**

DV 0.059*

0.073**

0.171***

NB 0.066*

0.081**

0.176***

-0.007"'

0.129**

Ghi chii: N, so mau thi nghifm; h, so lugng haplotype. (Cac dau •, **, *** la gia tri P ciia Chi^ the hien mirc dg khac bift tin cay, trong do * la 0.01<P<0.05; ** la 0.001<P<0.01 va *** la /'<0.00I. Ky hifu " la khac bift Ichong tin cay)

Cung nhu kit qui phan tich theo NJ, kit qua phan tich theo Nei (bang 2) mgt lin nira khang djnh khac biet di fruyin tin cay giQ-a cac viing bao tin (P**). Khi so sinh cip giira ong A.

cerana a Con Dio vi cic vimg khic, khac biet di fruyen li cao nhat (Gst= 0.117-1.176). Gii frj Gst= -0.007 chimg to ong A. cerana bao tin d Ding Van vi Nguyen Binh khdng co sai khac ve di truyin. Vi da dang di truyin, ci 5 quin thi ong bio tin ed da dang di truyen cao (Hd = 0.95).

Trong do, vimg ong Tiia Chiia cd da dang di truyen cao nhit (Hd=0.96) va thip nhat li vimg Con Dio (Hd=0.60).

Kit qui ciia 3 phuang phip phan tich NJ, khoing each di truyin ciia Tamura vi Nei vi khic bift di tmyin eiia Nei dya vio sy da hinh doan gen nghien cim ciia 39 mau A. cerana a 5 vimg bio ton cho thiy: 1) giira cac vimg bio tin cd khac bift di truyen tin cay; 2) giira hai viing ong Dong Van va Nguyen Binh khdng co khac bift vi di truyin; 3) trong moi vimg bao ton, da dang di truyen eiia ong A. cerana cao, ngoai trir vimg Cdn Dio ed da dang di truyen thap.

Dya vio da hinh ciia mtDNA, nhiiu nghien curu da chimg minh ring ong A. cerana d Hymalaya, Dong Duong, dong nam Trung Quic va Triiu Tien co da hinh ciia mtDNA cao. Day

863

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HOI NGHI KHOA HOC T O A N Q U O C VS SINH THAI V A TAI N G U Y E N SINH VAT LAN THCf BA

la CO sd giai thich tai sao A. cerana a nuac ta cd da dang di truyen cao khi phan tich dya vao da hinh doan COI ciia 39 miu A. cerana dai dien cho 5 vimg bao tdn ong. Tinh da hinh niy ciing da phan anh khac biet di truyen giira cic vimg bao tdn. Ket qua d bang 2 eho thay ong bio ton d Cdn Dio khac biet di truyin cao so vdi nhirng vimg khic (Gst= 0.117-1.176) cd tir 20 difm dgt biin die trung cho ong viing nay, trong khi do, ong d Tiia Chiia ed 11 diem vi d Ban Sen chi co 6 diim (hinh 1; B=bang 3). Vimg bio tin Ding Vin vi Nguyen Binh khdng ed dgt biin die trung cho timg vimg m i chi cd chung 14 dot bien die trung cho c i hai viing. Khdng co khic biet di tmyin giira hai vimg niy cd le do vj tri dia ly ciia chiing gin nhau, nen ong ed the di chuyen giira hai viing theo miia de lay thuc in, giao phdi...

Bdng 3 So dgt bien diem (DBD) dac trung giira cac viing va trong tirng viing bao ton

(vj tri cac dgt bien diem dac trung cho moi viing (xem hinh 1)

TC BS CD DV-NB

So DBD dac trimg giira cac viing bao ton

. 11 6 20 14

So DBD dac t n m g trong moi viing bao ton

13 7 1 7

Trong 5 vimg bao ton, ong Tua Chiia ed khoang each di truyen (d=0.0\3) va da dang di truyen (Hd=0.96) cao nhat vi cd tdi 13 diem dgt bien khi so sanh trinh tu ciia cac mau d vimg niy. Da dang di truyen cao nhit cua ong vimg Tiia Chiia cd the la do tiin hda ty nhien hoac la hau qua do con ngudi dua ong tir noi khac len nudi d vimg niy. Trii lai, chi cd 1 dot bien diem d viing bio tdn ong Cdn Dio, nen chiing ed chi s i da dang di truyin thip nhat (Hd=0.60). Quan the ong Cdn Dio cd da dang di truyin thap cd the do qui frinh tich bift cua chiing vdi dit lien trong thdi gian lau dii, dien tich viing khdng ldn, nen qui trinh ngiu phii giira cac thi hf cimg huyet thdng se lien tue xiy ra, din din ti If can huyit tang v i kem theo la suy giim vi da dang di truyen.

Khic biet di truyen cua ong A. cerana a 5 vimg bio ton d nude ta cd khi lien quan den phan loai, phit sinh chimg loai, phan bo... cua loai ong niy d Chau A. Tdng hgp cic cdng trinh nghien cuu ve chiing loai vi phan bo ciia ong A. cerana dya vao da hinh ve hinh thii, men va mtDNA tir nam 1955 din 1999 ciia cic tic gii tren the gidi, Hepbum va cs. da dua ra bin do phan b i cua 9 phin loii cua loii ong niy d Chau A (hinh 3). Dya vio kit qua phan tich cui cic tic gii nay cd the dy doan cd tdi 3 phan loii ong A. cerana a nude ta. Trong do, A. cerana cerana d cic tinh ddng bic giap Trung Quoc, A. c. indica a cic tinh cdn lai phia Bic tir miin Trung dd ra. Cdn d cac tinh miin Nam cd thi li A. c. indica hoac A.c.javana. Suy luan nay lien quan din kit qua trong nghien cuu cua chiing tdi. Vdi da dang di truyin (Hd=0.95) vi khic bift di truyen cao (Gst=0.l29) trong 5 vimg bao ton ong A. cerana, cd thi ong viing Dong Vin- Nguyen Binh li A. cerana cerana, vimg Tua Chiia v i Ban Sen thugc A. c. indica vi viing Con Dao ed the thugc A.c. indica hoac A. c. javana. Kit qua nay phin nio trimg hgp vdi kit luan cua Thii, eho rang ong A. cerana Ddng Van thugc phan loii A.c. cerana.

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HQI NGHI KHOA HOC T O A N QUOC V £ SINH THAI VA TAI N G U Y £ N SINH VAT LAN THCf BA

Tir ket qua ciia nghien ciru nay chimg toi xin dua ra mot so ggi y sau: 1) Ky thuat phan tir dya vao da hinh ve trinh ty tren COI ciia mtDNA trong nghien curu nay c6 thi dugc sir dung di nghien cim quan he di truyen giira cac loai, trong loai cua eac loii ong mat khac nhu A.

laboriasa, A. dorsata, A.florea, A. andreniformis dang dugc bao tdn d nude ta; 2) Viec dinh gia theo ky thuat niy can dugc tien hanh hang nam vi can bo sung cic mau ong A. cerana d cac vimg lan can quanh vimg bio ton de danh gii an toin nguon gen bao tdn; 3) Can tic dgng ky thuat tao giong de giim can huyet vi tang da dang sinh hgc ciia ong A. cerana a Cdn Dio; 4) Nen giu- ci hai vimg bio tdn Dong Van vi Nguyen Binh dii chiing khong co khac biet di truyen vi quan the niy mang mgt sd die tinh kinh te quan trgng dang dugc tien hinh khai thic de tao giong vi dua ra sin xuat.

Hinh 3: Phan loai ong A cerana vi phan bo ciia chiing d Chau A theo Hepburn vi cs. Chic in nghieng, ten phan loai; , vimg phan bo ciia cac phan loai theo cac tac gia niy; —, vimg phan bo

cua cac phan loai theo cac nghien ciru tir 1959-1999; ; gia dinh viing phan bo n i . KET LUAN

Vdi kit qua ciia nghien ciai nay, mgt s i kit luan co thi dugc nit ra nhu sau: 1) Khic bift di truyen va da dang di truyen cua 5 vimg ong A. cerana bio ton dugc nghien ciru thinh cong dya vao tinh da hinh vi trinh ty doan 1040bp fren gen COI ciia mtDNA ong thg ciia cac vimg bio ton nay; 2) Ong A. cerana a 5 vimg bao ton Tiia chiia (Dien Bien), Ding Vin (Hi Giang),

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HOI NGHI KHOA HOC T O A N Q U O C V ^ SINH THAI V A TAI N G U Y E N SINH VAT LAN THCf BA

Nguyen Binh (Cao Bing), Ban Sen (Van Ddn, Quing Ninh) va Phii Qude (Bi Rja-Vung Tiu) cd da dang di truyin cao v i khic biet di truyin tin cay; 3) Da dang d mdi vimg bio tin cao, ngoai trir d Cdn Dio - ong A. cerana cd da dang di truyin thip; 4) Do cac vimg bio tin co die thu rieng v i di truyin nen vifc chgn vimg bao tdn ong A. cerana nhu hifn nay li chinh xic.

TAI L i f U THAM KHAO

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4. Rozalski R. J., H. Sakurai, K. Tsuchida, 1996: Jap. J. Entomol., 64: 910-917.

5. Hepburn H. R., D. R. Smith, S. E. Radloff, G. W. Otis, 2001: Apidologie, 32: 3-23.

6. . Ward R. D., P. M. Grewe, 1994: Review in Fish Biology and Fisheries, 4: 300-325. _ 7. Smith D. R., L. Villafuerte, G. Otis, M. R. Palmer, 2000: Apidologie, 31:265-279.

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(Eds.), Evolution of genes and proteins, Sinauer Associates, Sunderland, MA, pp. 63-88.

10. Crozier R. H., Y. C. Crozier, 1993: Genetics, 133: 97-117

11. Garnery L., E. H. Mosshine, B. P. Oldroyd, J.-M. Cornuet, 1995: Moi. Ecol., 4: 465-471.

12. Diniz-Filho JAF, S. Fuchs, M. C. Arias, 1999: Heredity, 83: 671-680.

13. Songram O., S. Sittipraneed, S. Klinbunga, 2006: Biochemical Genetics, 44: 256-269.

14. Pham Hong Thai, 2008: Nghien ciru mgt s i die diim hmh thii, sinh hgc phan hr (ADN ty the) cua eac quan the ong ndi A. cerana F. phan bo d Vift Nam va hudng de xuat sir dung ngudn gen tren vio cdng tic chgn tao gidng ong mat cua nude ta. Luan van Tien sy.

15. Garnery L., M. SoUgnac, G. Celebrano, J. M. Cornuet, 1993: Experientia, 49: 1016-1021.

16. Kumar S., K, Tamura, M. Nei, 2004: MEGA3: Briefings m Bioinformatics, 5: 150-163.

17. Rozars J., J. Sanchez-Debarrio, X. Messeguer, R. Rozars, 2003: Bioinformatics, 19:

2496-2497.

.18. Nei M., 1987: Molecular Evolutionary Genetics. Columbia University Press, New York: 512.

19. Tamura K , M. Nei, 1993: Moi. Biol. Evol., 10: 512±526.

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25. Page R. E., H. H. Laidlaw, 1985; Bee Worid, 66(2): 63-72.

26. Oldroyd B. P., G. J. Thompson, 2007: Advances in Insect Physiology, 33: 1-49.

GENETIC D I F F E R E N T I A T I O N O F CONSERVED HONEYBEE APIS CERANA IN VIENAM: EVIDENCE FROM COI SEQUENCE POLYMORPHISM OF MTDNA*

LE QUANG TRUNG, NGUYEN DUC LAM, DINH THI DAN NGUYEN TUONG VAN, VU THI HIEN NGUYEN NGOC VUNG, PHUNG HUU CHINH SUMMARY

Evaluation of genetic differentiation between conserved populations of A. cerana and genetic diversity within each such population is one of the important works to reveal effectiveness of the in-situ conservation approach for sustainable use of honeybee resources in Vietnam. Recently, polymorphisms of different parts on mtDNA have been widely employed for this purpose. In this research, primers were designed using different COI-allele sequences of A. cerana and A. dorsata, which were published on the GeneBank. Two primers FTr_Acd va RTr_Acd were successfully applied to PCR-amplify with total DNA of 39 workers from 5 different^, cerana-conserved regions in Dong Van (Ha Giang), Nguyen Binh (Cao Bang), Ban Sen (Van Don, Quang Ninh) and Con Dao (Ba Ria - Vung Tau), and one A. dorsata from Moc Chau (Son La) used as control. Results of amplification and sequencing revealed 1040bp-PCR products for all samples. Polymorphic analyses of those sequences, based on Neighbour Joining, Tamura vi Nei (1993) and Nei (1973), indicated that genetic differentiation among and between populations was significant (P of Gst from * to *** of significance), except between Dong Van- Nguyen Binh showing no genetic differentiation ("'for P of Gst). This also pointed out that genetic diversity within each population was high (Hd = 0.78-0.96) except for Con Dao, whose A. cerana population is low at genetic diversity (Hd=0.60). Significance of genetic differentiation revealed accuracy in location of 4 conserved A. cerana regions. The approach on COI sequence analysis can be applied for research on genetic relationship at mter- and intra- species level of other Apis species that have been in conservation in Vietnam.

Cdc tdc gid xin chan thanh cam an tap the cdn bp cua Trung tdm Nghien ciru vd Phat trien Ong vd Vi?n Cong ngh? sinh hpc dd tgo dieu ki?n thuc hi^n thdnh cong nghien cicu ndy. Nghiin cicu ndy ndm trong noi dung ciia de tdi 'Luu giu: Insitu cdc loai ong Viet Nam nam 2009 " do Bp

^ong nghiep vd Phat triin nong thon vd Bp Khoa hpc vd Cong nghe cdp kinh phi vd quan ly.

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