TAP CHI KHQA HQC BAI HQC V A N LANG Trirong T h i Quang
PHAN NHOJM MANG CAU DAI {ANNONA SQUAMOSA L.) VUNG NUI BA DEN, TINH TAY NINH
DlTA TREN GENE matK VA HINH THAI HOA
CLASSIFICATION OF CUSTARD APPLE (ANNONA SQUAMOSA L.) INBA DEN MOUNTAIN AREA. TAY NINH PROVINCE
BASED ON GENE MATK AND SHAPE FLOWER
TRUONG THE QUANG^'' TdM TAT: Gidi trinh tu gene matK cua mudi mdu Id non mang cdu dai (Annona squamosa L.) d vung nui Bd Den, tinh Tdy Ninh. Lfng dung ky thugt ddu phdn tu matK thupc hi gene lue lgp de xdc dinh moi quan hi di truyen giiia cdc lodi mdng cdu dai Bd Den. Xdy dung cdy phdt sinh lodi. phdn logi cdc nhom lodi mdng cdu dai Bd Den dya trin trinh tu gene matK vd dgc diem hinh thdi hoa.
Tic khda: mang cdu dai, matK, niti Bd Den.
ABSTRACT: Gene matK of ten custard apple young leaves (Annona squamosa h.) in Ba Den mountain area, Tay Ninh province have been sequenced. Application molecular markers gene matK of ehloroplast genome, genetic relationship between species of custard apple Ba Den have been defined. Based on gene matK and shape flower, phylogenetic tree and classification species of custard apple Ba Den have been developed.
Keywords: Custard apple, matK, Ba Den mountain.
1. DAT VAN DE va khoang chdt. Ngodi ra, mang cdu cdn cd Cay mang cdu cd nguon gdc tu Chdu mgt huang vj dgc bidt khiln nhieu ngudi ira My. Mang cdu ta {Annona squamosa L.) cd thich, cd do nggt cao, vi chua khdng lgt, cd ten tilng Anh: Custard apple, Sugar apple; huong thom.
tdn tilng Phdp: Pomme cannelle. Tir thd ky Trong cdng tdc bao tdn vd sd dyng ben XVI, cdy mang cdu da dugc du nhap vao vung tdi nguyen di truydn thye vgt, viec danh nhilu nude nhiet ddi do tinh thich nghi d gid quy gene la cdng dogn vd ciing quan cac vung nhiet ddi, a nhiet ddi va da mang trgng, khdng chi phyc vy cho vide xac djnh lai nhilu gid tri kinh tl. Mang cdu ta d phia cdc gidng, lodi khac nhau ma cdn nhdm tim nam nude ta thudng co hai loai la mang cdu hilu mli quan he ve di truyen giiia cac gidng, dai va mdng cdu bd. Tuy nhien, mdng cau loai de bao tdn da dang ngudn gene. Sy phdt bd khdng cd nhilu uu diem ve phdm chdt trien manh me cua cac phuang phap va ky va gia trj kinh t l nen phdn ldn ngudi dan thugt trong ITnh vyc sinh hgc phdn tu da tgo trdng mang cau dai, la loai qua giau sinh td ra cdc cdng cu hiiu hieu vd nhanh chong '"'TS. Truong Bai hgc Van Lang, Email [email protected]
119
TAP CHl KHOA HQC BAI HQC V A N LANG dugc img dung trong nghien cdu bdo tdn da dang sinh hpc. U'u till cua cdc ky thudt phdn td la cd khd nang xdc djnh duge sy da dang d mdc dp gene, tgo ca sd de ddnh gia ve gia trj bdo tdn eda loai vd qudn thd. Trong dd, ma vach dya tren trinh ty DNA (DNA Barcode) ra ddi da hda hen mgt tuong lai mdi, noi ma con ngudi c6 thd cap nhdt nhanh ehdng cac thdng tin nhu ten, thugc tinh sinh hpc,... cua bdt cu loai sinh vdt ndo ddn trdi ddt. Mpt cdng cu him hi?u trong viec dinh danh thyc vdt khi cac dQ' Ii?u hinh thdi bi thilu hoac bat lyc trong viec so sanh dd phan bidt cac loai trong ciing mdt gidng. Chinh vi vay, viec tgo lap CO sd dii lieu DNA ciia cac gidng, lodi de ddng ky d ngan hang gene the gidi vd tdi nguydn di truyen thyc vgt dgc hiiu cua Vidt Nam ndi chung vd mang cdu ndi rieng dang la van de rat quan trgng, mang tinh khoa hpc vd thyc tidn cao. Trong nhiing ndm gdn day, ngudi ddn chuOng trdng cac gidng cay mdi dupe lai tgo bode du nhdp tu nude ngoai vao.
Chinh nhiing vdn d l ndy da va dang lam giam sut mpt sd gidng quy, dde him cua dja phuang, trong dd cd cay mdng cdu dai viing mii Bd Den thuOc tinh Tdy Ninh. Do do, vide xac dinh moi quan he di truydn vd phdn djnh cac nhdm loai mang cdu dai dang trdng tgi viing nui Bd Den thudc tinh Tdy Ninh dya trdn trinh ty gene matK va dgc diem hinh thai hoa la can thiet, ddy la ca sd khoa hpc ban dau de xdy dung bdn dd gene cho gidng mang cdu dai {Annona squamosa L.) dac hii'u tai dia phuang nay trong viec bao tdn nguIn gene, xdy dyng thuang hidu sdn phdm thdng qua chi ddn dia ly va bao hp thuang hieu cua san phdm, cung nhu ung dyng trong nghien cdu chpn gidng, lai tao gidng mdi khdng benh vd cd chat lugng cao.
Hmh 1 Cay mang cau dai trong ta vung nui Ba Den, tinh Tay Ninh [8]
Trong hd thdng hpc thyc vgt gdn day, gene matK ndi len nhu la mot gene vd gid bdi ddu hieu phat sinh chung loai cua nd cao so vdi eac gene khdc dugc dung trong ITnh vye nay. Gene matK (md hda cho maturase K) dugc phdt hien ddu tien tren cay thudc la {Nicotiana Tabacum) khi gidi trinh tu gene tmK ma hda cho tRNALys (UUU) cua lyc lap [2], [6].
Gene matK cua Annona squamosa dai khodng 802 bp, tuong dng vdi khoang 267 amino acid trong protein [5]. Ndm giiia hai exon d ddu 5' va 3 ' cua trnK, tRNALys trong vung ban sao don Idn cua bd gene lyc Igp d hdu hit cdc lodi thuc vat ddt (Hinh 2).
Gene matK ndi bdt len trong sd cac gene lyc Igp dupe diing trong phdn tich he thdng hpc thyc vat theo kilu rieng va nhip tiln hda cua nd. Sy biin ddi nucleotide tinh trdn mdi vi tri trong matK cao gdp 3 Idn trong rbcL (tiiu phdn ldn cua RUBISCO), va tdc dp thay the amino acid cao gap 6 Idn cua rbcL. Cung vdi tdc dd thay thi amino
TAP CHl KHOA HOC BAI HOC V A N LANG Truwng The Quang
acid cao trong matK la ti Id thay thi gan nhu bdng nhau giira ca ba vj tri trong moi codon. I 5 c dp thay thi cao Idm gia tang sd didm parsimony va tin hidu phat sinh chung lodi manh, ndn nd cd the dugc su dung de phdn biet Ijch su tien hda d cac muc phdn logi khac nhau. Gia trj ve thdng tin phdt sinh chirng loai tgo ra nhd matK Idm cho no trd thanh gene cyc ky cd gid trj trong cac nghien ciiu he thdng va tien hda nhanh ehdng [1]. Tdc dp thay the amino acid nhanh trong gene matK hdu nhu la do cd tdc dp thay thi phdn bd ddng diu cho cd ba vj tri trong codon trong khi phdn ldn cdc gene ma hda protein khac lgi cd xu hudng thay thi nucleotide d vi tri thd ba. Chang han, tdc dp thay the d 3 vj tri trong codon ciia matK ldn lugt la 62%, 57% va 66% khi
so sdnh cdc bd thyc vat hat kin vdi nhau [3], vd Idn lugt Id 32%, 28% vd 39% trong hg Lan (Orchidaceae) [9]. Cdc d^t biin chen hay mdt (ggi tdt la xda chen - Indel) thudng xdy ra trong matK, mac du cac xda chen chu ydu xdy ra vdi bgi sd cua ba, duy tri khung dgc md. Tdc do thay the nhanh ciing vdi su hidm hien dien cac xda chen lam thay ddi khung dgc vd mgt vdi trudng hpp codon kit thuc xudt hidn sdm.
Thdng tin trinh ty td mdt minh gene matK dd cd thi tgo ra kit qua phdt sinh chung loai mgnh nhu khi dyng bang cdc bg dii lieu gdm td 2 den 11 gene khdc nhau ket hop. Han niia, thdng tin phdn td td matK cung dupe sd dyng d l phdn tich cac quan he phat sinh chung loai d cdc nhdm phdn logi td thdp din cao [3].
MSlcAon^j Lti'itKiimul
Hinh 2. Gene matK, viing Reverse Transcriptase (RT) cua matK ORF, mien domain X va hai viing exon trnK
0 nude ta hidn chua cd cac nghidn cuu ve quan he di truyen cua cdc loai mang cdu (Annona squamosa L.) dya trdn cac ddu phan tu thupc he gene luc lap hodc he gene hong nhan. CJ nude ngoai da cd cdc cdng trinh nghidn cdu ve Annona squamosa nhu
"Md vgch DNA cua mpt sd lodi thupc hg na (Annona) " cua Nerea Larranaga va Jose I. Hormaza (2015) [5]; "Da dgng sinh hoc ciia mpt sd lodi thupc hg Na (Annona) vd hinh thdi qud dua theo cdc kiiu gene da chgn" cua Hirdayesh Anuragi vd cpng sy (2016) [4].
2. MUC TIEU VA NOI DUNG NGHIEN CUU
2.1. Muc tiSu nghidn cuu
Gidi trinh ty gene matK cua mudi mdu la non mdng cau dai dang trdng d vimg nili Ba Den, tinh Tdy Ninh. IJng dung ky thu^t ddu phdn td matK thuOc hd gene luc Igp dh xac djnh mdi quan he di truyen gida cac loai Annona squamosa L.
Ba Den. Xdy dung cdy phat sinh loai, phan djnh cdc nhdm lodi Annona squamosa L. Bd Den dua tren trinh ty gene matK va dac diem hinh thai hoa.
Nghidn cdu dugc thuc hien vao ndm 2017
TAP CH! KHQA HOC BAI HOC V A N LANG tgi Phdng thi nghiem Sinh hpc phdn td, Vidn Phap y Thdnh phd Hd Chi Minh.
2.2. Npi dung nghien cuu
Ldy mdu la non mang cdu dai tgi cac xa Thanh Tan, Tdn Binh, Phudc Binh, Phudc Tdn, Sudi Dd thudc khu vyc chdn nui Bd Den, tinh Tdy Ninh;
Tach chiit va thu nhan DNA tdng sd bdng phuong phap CTAB cd hieu chinh;
Thilt k l va tdng hgp cap mdi 2. IF va 5R dung dk khulch dai vung gene matK;
Khulch dai vung gene matK bang ky thudt PCR vdi cap mdi 2.IF va 5R;
Didn di tren gel argarose 1% de ddnh gia sdn phdm PCR;
So sdnh brinb ty gene matK vdi co sd du: lieu GenBank bdng phan mem tryc tuyIn Blastn d l djnh danh loai mang cdu dai vung nui Bd den Id Annona squamosa L. Ba Den;
Thiet lgp ma tran ty Id tuang ddng vd xdy dung cdy phat sinh loai ciia 10 mdu
Annona squamosa L. Bd Den dua trdn trinh tu gene matK va dgc dilm hinh thdi hoa;
Phdn djnh cdc nhom loai Annona squamosa L. Bd Den dya tren trinh ty gene matK va dgc dilm hinh thai hoa.
3. PHUONG PHAP NGHIEN Ctl'U 3.1. Tach chiit DNA t i n g sd
Cdc mdu la non mang cdu thu thap sd dugc tdch DNA tdng sd bang bg kit CTAB (HiMedia Lab.). Sau dd, DNA tdng sd se dugc kiem tra dp tinh sgch bdng didn di tren gel agarose 1%. Mdu DNA dat yeu cau se dugc bdo quan trong dieu kidn Ignh sdu (- 20 °C) eho cac nghidn cdu vd sau.
3.2. Khulch dai vung matK
Cap mdi dung khuech dgi viing gene matK dupe thiet ke bang phdn mem DNAMAN phien ban 9 (Lennon Corporation, 2017) va dupe tdng hgp tai Phdng thi nghiem Sinh hpc phdn td, Vien Phdp y Thanh phd Hd Chi Minh (Bdng 1).
Bang 1. Cap moi dupe sii dung trong ph^n iing khuyech dai vOng matK [8]
Trinh tu: moi theo chieu 5'- 3' Tm CO
Chilu phan ung
Chieu ddi san phSm du'kien
(bp) 2.1F CCTATCCATCTGGAAATCTTAG
5R G T T C T A G C A C A A G A A A G T C G
5S,2 Xuoi 56,7 Ngupc
Thanh phan hdn hpp phan dng va chu trinh nhiet cua phan dng PCR khuech dai vung gene matK sd dyng cap mdi 2.IF vd 5R (Bdng 2, 3).
Bang 2. Cac thanh ph^n c6 trong phan ung khulch d?i viing gene matK [8]
STT Thdnh phan The tich (jil)
1 Taq DNA pol 2x - preMix 2 M6i2.1F(5nM) 3 Mdi5R(5pM) 5 DNA khuon
12,5 1,0 1,0 9,5 Dieu chinh
Tong the tich 25,0
i i r
TAP CHl KHOA HQC BAI HQC VAN LANG Truong The Quang
Bang 3. Chu ky nhifit khuSch dai vung gene matK [8]
Nhigt 69 (°C) Thdi gian C h u k y Tifin bien tinh
Bien tinh B5t c5p Keo dai Ket thuc
94 94 53 72 72
3phiit 30 giay 40 gi§y 40 giay 5 phut
1 30 30 30 1 3.3. Dien di san phdm PCR
Mdu sdn phdm PCR dugc bd sung thudc nhugm ethidium bromide, dung djch ddm loading buffer va cho chgy di^n di tren gel agarose 1% vdi hieu didn thi 120 V, thdi gian tir 30 din 60 phdt. Quan sat cdc band DNA trdn ban dpc gel UV vdi budc song 365 nm.
3.4. Gidi trinh t y sdn ph^m PCR San phdm PCR sau khi thu nhgn se dugc gdi di giai trinh ty theo chieu xudi vd chilu ngupc bdng he thdng ty ddng 3130.
Thi tich san phdm PCR ddng de gidi trinh ty la 10 1^1. Cap mdi 2.IF va 5R dupe sd dung d l giai trinh ty. Mdi phai dugc pha trong nude cdt hai ldn khd ion, khdng cd mudi, EDTA hay bdt ky nhiem tgp khac.
Ndng do cua mdi mdi Id 10 \iM vdi thi tich tii thilu la 5 \i\ cho moi mdu giai trinh ty.
3.5. Hieu chinh trinh tu-
Trinh ty sau khi dupe xac djnh bang he thing ty dpng 3130 chua thi sd dung ngay cho vide phdn tich. Vide dpc base ty dpng do he thing thye hidn cd mdt ty le sai sdt nhdt djnh tuy theo phuong phap va logi mdy sd dung. Sy sai sdt nay xay ra bdi cudng dp tin hieu huynh quang thu dugc khdng phai liic nao cung ro rang. Khodng each khdng ddng diu giiia cdc mui tin hidu cung nhu sy ehdng ldp cae tin hidu dan den vide mdy tinh nhan vd hien thi sai ket qua.
Hieu chinh trinh ty bdng each loai bd
nhung tin hieu khdng chinh xac d hai dau trinh ty va kiem tra cac sai lech giiia hai kit qua gidi trinh ty.
3.6. So sdnh vdi co' sd dii- liSu GenBank Sau khi hidu chinh cac sai lech, trinh ty lidn ung (Consensus Sequence) dupe rut ra td hai kdt qua giai trinh ty va kilm tra cac sai lech. Trinh ty lidn ung la trinh ty chinh xdc cho dogn DNA khao sat. Tuy nhien, kdt qud nay cdn dupe kiem tra mgt ldn niia tren CO sd du lieu nucleotide nhu GenBank bdng cdng cy BLAST. Ndu cd cdc sai lech giiia trinh ty trdn ca sd du lieu va trinh tu truy van thi cdn dugc xem xet vd kidm tra Idn nua de xdc nhgn ket qud gidi trinh ty.
Ngoai ra, viec thyc hien BLAST cdn giiip kilm tra sy nhidm mdu va danh gia qua trinh thu nhgn vd bdo quan mdu.
3.7. Xay du-ng cay phat sinh loai Xdy dyng cay phdt sinh loai bang phan mim DNAMAN phien ban 9. Sdp hdng nhilu trinh tu bdng thudt toan ClustalW (Feng - Doolittle and Thompson). Thidt lap ma tran tuang ddng va ma trdn khoang each theo md hinh Kimura (Kimura, 1980, J. Moi. Evol 16: 111-120). Phdn tich bootstrap 10.000 ldn cho gia trj dd tin cdy cua cay phdt sinh loai.
Cdy Phylogenetic dugc thilt lap td ma tran khodng each theo thuat toan Neighbor - Joining (Saitou vd Nei, 1987, Moi. Biol, Ecol.4: 406-425) [7].
TAP CHI KHOA HQC BAI HQC V A N LANG 4. KET QUA VA THAO LUAN 4.L Thu thap va xii' ly mau
Mdu la non mSng cdu dupe thu thdp d cae xd thupc khu vyc chan nui Ba Den.
Dupe dinh danh va phdn logi so bd theo hinh thai hoa (Bang 4). Mdu dugc xu Iy vd bao qudn d - 20 "C cho vide tach chiet DNA tdng sd.
STT 1
2
3
4 5
Biing 4. S6 luxmg mau la va hinh th^i hoa mang cau Xa, thanh pho (huy^n)
Xa Thanh Tan, thanh pho Tay Ninh
Xa Tiin Binh, thanh plio Tay Ninh
Xa Phudc Tan, huy|n Duong Mmh Chau
Xa Suoi Da, huyen Duang Minh Chau Xa Phuoc Binh, huyen Duong Minh Chiiu
T5ng cong:
So lirgng mau
01 01 01 01 01 01 01 01 01 01 10
,[8]
Ky higu
XI T l X2 T2 X3 T5 X4 T6 T3 T4
Hinh thai hoa Xoan Th§ng Xoan Thang Xo5n Thang
Thang Thang Thang
a) Hoa xoan
Hinh 3. Hinh thai 4.2. Tach DNA tdng so
Trong nghidn cdu'ndy, DNA tdng sd ciia cdc mdu dugc tdch bang phuang phdp CTAB theo Doyle (1987) cd hieu chinh chinh phu hpp vdi tinh trang thyc t l cua mdu. K i t qud dugc kilm tra bdng dien di tren gel agarose 1%. Ket qua didn di trdn gel agarose cho thay cdc band DNA thu dugc ciia cdc mdu mang cau cd vet sdng keo dai, dieu dd chung
b) Hoa thang hoa mSng clu [8]
td vdn cdn nhieu RNA ehua dupe loai bd khdi dich chiet DNA.
4.3. Khuech dai gene matK bdng ky thuat PCR
Tien hdnh khuech dgi gene matK vdi chu trinh nhiet nhu dd trinh bdy d Bang 3.
Kit qua didn di san phdm PCR cho thdy d mdu T3, T4 va T6 khdng cho san phdm PCR nhu mong mudn. M l u X I , T2 vd T5 cd ndng dp san phdm PCR thdp. Do do.
TAP CHi KHOA HQC BAI HQC V A N LANG Tru-Q-ng The Quang cdn hieu chinh bdng each tang ndng dp
DNA khudn.
Dd khdc phyc, thyc hi?n phdn dng PCR lan hai cho cdc mdu chua dat yeu cdu vdi DNA khudn thay ddi la 1,2 pi vdi cdc mlu XI, T2 va T5 va 1,5 |j,l ddi vdi cac
mdu T3, T4 vd T6. Ket qua chay PCR Idn 2 da khulch dgi thanh cdng gene matK, cho sdn pham dung nhu mong dgi. Cdc band sdn phdm rd, dung kich thudc nen cd thi su dung gidi trinh ty (Hinh 4).
'•^:. '^'•^'^f^w^^mi^^^^^
Hinh 4. Ket qua dien di san pham PCR gene matK ISn 2[8]
4.4. K e t q u d giai t r i n h t y vd hidu c h i n h trinh t y
Ket qua giai trinh tu gene matK, tdt cd 10 mdu mang cau ddu cho kit qud giai trinh ty tdt cho cd hai mach mdi xudi vd mdi nguge. Chdng td cap mdi 2.IF va 5R rdt hieu qua trong viec khuech dai va gidi trinh ty gene matK. Trinh ty gene matK cua 10 mdu mdng cdu cd kich thudc tu 756 bp din 844 bp [8].
Bang 5. Cac U
4.5. So sdnh trinh t y cua mau vdi cff sd du'lieu GenBank
Kiem tra sa bd bdng cdng cu BLAST tren ngan hang GenBank eho thdy, tdt cd 10 trinh tu ndi tren diu thudc cdc loai mang cau Annona squamosa L. cd trong GenBank vdi mdc dp bao phu 99% vd mdc do tuang ddng 100% (Bdng 5). Dilu ndy cho thdy, mau vat thu thdp ngoai thyc dja khdng bj ldn tgp cac mdu khdc, qud trinh bdo qudn mdu tdt dat dp tin cay cao.
Annona squamosa L. trSn GenBank c6 dO tucmg dong cao vdri mlu Ten loai Quoc gia Accesion Number A. squamosa L.
A. squamosa L.
A. squamosa L.
A. squamosa L.
Tmng Quoc Trung QuIc An DO Tay Ban Nha
EU715Q64.I KT452823.1 JNH 4737.1 KM68854.I
Vide so sdnh vdi co sd du lieu tren GenBank nham myc dich cho mgt ket qua tham khao vdi nhdm lodi tuong dong nhdt vdi trinh tu truy vdn. Vdi nhQng trudng hpp BLAST cd dp bao phu 100% va dp tuang ddng 99% cd the suy dupe ten lodi.
Bdi ket qua BLAST hien thj trinh tu tuang ddng nhat ma tren GenBank hien cd va vdi nhii'ng trinh tu d mu'c dp dudi lodi, nen dya tren vung trinh ty gene matK cd thd ket luan 10 mdu mang cdu ndi Bd Den, tinh Tdy Ninh la loai Annona
TAP CHI KHOA HQC PAI HQC VAN LANG So 08/2018 squamosa L. Ba Den, Viet Nam, chua
dugc cdng bd vd cap nhat vao co sd d\t lipu GenBank tren the gidi.
4.6. Xdy dung cdy phdt sinh lodi
Ung dung phdn mim DNAMAN phien ban 9. Sdp hang 10 trinh tu gene matK ciia Annona squamosa L. Bd Den bang thuat todn ClustalW. Kdt qua, thilt ldp dugc ma trdn khodng each (Bang 6) theo md hinh Kimura.
Bang 6. Ma trSn khoang each ciia 10 trinh ty gene matK A. squamosa L. Ba Ben [8]
T l T2 T3 T4 T5 T6 XI X2 X3 X4
TI 0 0.003 0.006 0.004 0.003 0.004 0.077 0.129 0.077 0.077
T2
0 0.004 0.004 0.003 0.004 0.076 0.129 0.076 0.076
T3
0 0.004 0.003 0.004 0.077 0.129 0.077 0.077
T4
0 0.004 0.004 0.075 0.130 0.075 0.075
TS
0 O.O03 0.073 0.128 0.073 0.073
T6
0 0.074 0.127 0.074 0.074
XI
0 0.092 0.000 0.000
X2
0 0.110 0.112
X3 X4
0 0.001 0 Cay Phylogenetic (Hinh 5) dugc thilt
lap td ma trdn khodng each theo thudt todn Neighbor - Joining. Phdn tich bootstrap
10.000 lan cho gid trj dp tin cay cila cdy phat sinh loai.
TS
Hinh 5. Cay Phylogenetic cua A, squamosa L. Ba Den theo trinh ty gene matK [8]
TAP CHI KHQA HQC BAI HQC V A N LANG Tru-ffng The Quang 4.7. Phan nhom mang cau dai vung nui
Ba Den theo gene matK
Phan tich cay Phylogenetic theo gene matK cd the chia Annona squamosa L. Ba Den thanh ba nhdm dudi lodi: Nhdm 1:
Annona squamosa L. Bd Den hoa xoan gdm ba mdu X I , X3 va X4; Nhdm 2:
Annona squamosa L. Bd Den hoa xoan.
Bang 7. Ty !§ tuang ding, khoang each tuong dong cua
gidng Thai Lan gdm mdt miu X2; Nhdm 3:
Annona squamosa L. Ba Den hoa thang gdm 6 mdu T l , T2, T3, T4, T5 va T6.
Ty Id tuong ddng (%) vd khoang each tuong ddng cua ba nhdm Annona squamosa L. Bd Den dugc neu trong Bdng 7.
i nhom A squamosa L. Ba Den [8]
Nhom A.
sqtiiiinosa L.
Ba Den Nh6m 1 Nhom 2 Nhom 3
Nh6m 1 Tuang dOng Khoang each
100% 0.000 90% 0.102 92% 0.073
Nhom 2 Tucmg dOng Khoang each
90% 0.102 100% 0.000 87% 0.127
Nh6m 3 Tirong d6ng Khoang each
92% 0 073 87% 0.127 100% 0.000 5. KET L U ^ N VA KIEN NGHI
Tdch chiet va thu nhan dupe DNA tdng so CLia 10 mdu mang cdu dai (Annona squamosa L.) vung ndi Ba Den, tinh Tay Ninh bdng phuang phdp CTAB cd mdt sd hidu chinh;
Khulch dai dupe vung gene matK, kdt qua thu dupe sdn pham dung kich thudc, khdng tgp nhidm;
Cap mdi 2.IF va 5R cd hieu qua trong viec giai trinh tu hai chieu gene matK vdi todn bd 10 mau Annona squamosa L.
Bd Den;
Sau khi so sdnh trinh tu gene matK vdi CO sd dd lieu GenBank, cho thdy tdt ca 10 trinh tu gene matK deu thudc loai Annona squamosa L. vdi dp bao phu 100% vd dp tuang ddng 99%. Dieu nay cho thdy, mau vdt thu thap ngoai thyc dja khdng bi ldn tap cac mdu khac, qua trinh bao quan mdu tdt v d d a t d d t i n c ^ y c a o ;
Xdy dyng dugc cay phat sinh lodi ciia 10 mdu Annona squamosa L. Ba Den dya tren trinh tu gene matK;
Lan dau tien giai trinh ty gene matK thanh cdng cho ba nhom lodi dugc dinh danh so bg hinh thdi gdm Annona squamosa L. hoa xoan (XI, X3 va X4), Annona squamosa L. hoa xoan, gidng Thai (X2); Annona squamosa L. hoa thang (Tl, T 2 , T 3 , T 4 , T 5 v d T 6 ) .
Kiin nghi:
Thu thap them mdu mang cdu (Annona squamosa) a cac vung phy can nui Ba Den, tinh Tdy Ninh dd nghien cdu da dgng di truyen cua loai nay;
Can sd dung thdm mdt sd marker trinh tu cua cac vung khac nhu rpoB, rbcL, accD,... de cd the djnh danh loai, dudi lodi va xay dyng he thdng DNA barcode cho cac loai Annona squamosa L. Viet Nam.
TAP CHi KHOA HQC BAI HQC VAN LANG s l 08/2018 TAI LIEU THAM KHAO
1. Barthet, Michelle M.; Hilu, Khidirw (2007), Expression of matk: functional and evolutionary implications.
2. Daniell H., Lee S. B., Grevich J., Saski C , Quesada Vargas T., Guda C. B., Tomkins J., Jansen R. K. ^006), Complete ehloroplast genome sequences of Solanum bulbocastanum, Solanum lycopersicum and comparative analyses with other Solanaceae genomes. Theoretical and Applied Genetics 112.
3. Hilu K. W., T. Borsch, MuIIer K., Soltis D. E., V. Savolainen MW (2003), Angiosperm phytogeny based on matK sequence information, American Journal of Botany 90.
4. Hirdayesh Anuragi, Haresh L. Dhaduk, Sushi! Kumar, Jitendra J. Dhruve, Mithil J.
Parekh, Amar A. Sakure (2016), Molecular diversity of Annona species and proximate fruit composition of selected genotypes, 3 Biotech (2016) 6:204, Springeriink.com.
5. Nerea Larranaga, Jose I. Hormaza (2015), DNA barcodlng of perennial fruit tree species of agronomic interest in the genus Annona (Annonaceae), Frontiers in Plant Science, Volume 6.
6. Steane D. A. (2005), Complete nucleotide sequence ofthe ehloroplast genome from the Tasmanian blue gum. Eucalyptus globulus (Myrtaceae), DNA Research 12.
7. Truang Thi Quang (2017), Tin sinh hgc (Biolnformatics), Gido trinh luu hanh ndi bp, Trudng Dai hpc Van Lang.
8. Trirong The Quang, Vd Trdn Bdo Vy (2017), Xac dinh quan he di truyen vd phdn bo dia ly cdc lodi mang cdu dai (Annona squamosa L.) viing niii Bd Den, tinh Tdy Ninh dya tren trinh ty vung gene luc lgp matK, Trudng Dgi hpc Vdn Lang.
9. Whitten et al. (2000), Subtribal and generic relationships of Maxillarieae (Orchidaceae) with emphasis on Stanhopeinae: combined molecular evidence, American Journal of Botany 87(12).
Ngaynhdnbai: 15/01/2018. Ngdy bidn tap xong: 17/01/2018. Duyet dang: 17/3/2018.