KHOA HOC CdNG NGHE
IVHAIV DIEIV PHAIM TU MOT SO LOAI LAIXI HAI [PaphiapedUunii DAC HLTU TAI VIET IXIAIVI
Vu Thi Huy^n TrangS Vu Quoc Lu|^^ L6 Tlii Ngpc Di6p\
Nguyen Thanh Di^mS Nguyin Tli^nh C6ng\ Um Phuong NamS Nguyen Htm Thudn AnhS Tran Duy Duomg^, Tran Hgp\ T r ^ Homing Dung^
TOMTAT
Lan Hai Papbiopedilam dac him Viet Nam la nhom lan quy hiem, co nguy co tuySt chiing cao nen can c6 bien phap bao ton. Trong khi da sd cac cay lan Hai dtroc mua ban thuong mai thuong la cay con, cay chua tnrong thanh, cay khong co hoa vi the can phai co phuong phap nhan dien nhanh va chinh xac. Hien nay phuong phap ma vach DNA la mot phuong phap hieu qua, duoc ung dung ngay cang pho bien. Trong nghien cuu nay de nhan dien cac loai lan Hai da khao sat 6 vimg gene la ITS, maK. tmL. rpoB, rpoCl, tmK- psbA tren 23 mSu thuoc 8 loai lan Hai dac hiiu Viet Nam. Viec khuech dai cac vimg trinh tu ITS, matK, tniL, ipdB, rp(Cl deu thanh cong voi hiSu suat tuyet ddi 100%. Cac trinh tu lan Hai dac hiru Viet Nam trong nghien cuu du^c dang Id ma sd trinh tu, dong thoi gop phan vao thu vien trinh tu Gen the gioi. Ket qua cua nghien cuu da d4 xuat trinh tu ket hop ITS va maS^ cho tiem nang cao nhat trong vi§c nhan dien 6 loai lan Hai dac huu trong nghien cuu, gdm P. delenatii, P. bangianum, P. helenae, F. x dalatense, P. grabixianum, P. vietnamense. Trinh tu ma vach nay cd the ap dung de nhan dien quan the lan Hai Viet Nam phue vu cho viec quan li va bao ton cac loai lan Hai co gia tri.
Tukhoa: Papbiopedilum, loai dac hihi, nhan dien phan tu, ITS, matK. tmL
I.GiOITHP
Viet Nam la niroc co moi tnrdng sinh thai da dang thich hop voi nhi^u loai cay, trong do co lan Hai. Lan Hai dac hiru la lan chi t6n tai va phan bo cr mot khu vuc nhat dinh vi vay dupe ua chuong vi tinh quy hiem. Di6u nay dan d^n viec buon ban trai phep lam cho lan Hai ngay cang gan voi nguy ca tuyet chung. De bao ton can co phuong phap nhSn dien nhanh va chinh xac cay ngay ca trong giai doan chua CO hoa.
Phuong phap ma vach DNA sir dung mot doan DNA ngan trong bo gen nhan, luc lap hoac ty the nhu la mot chi thi de nhan dien mpt nhom ca the dac tnmg vm do chinh xac a miic nucleotide. Ngay sau khi khai niem ma vach DNA ra doi, nhi^u cong trinh tren the gioi da tap trung vao viec thiet lap bp dir lieu DNA cho sinh vat ban dia. Nhieu cong trinh duoc img dung tren lan Dendrohium dua vao cac vimg trinh tir/JsM-£r7?H (Yao etal., 2009), madCva rfid. (Asahina et al, 2010). Tr6n nhom dia lan Cymbidium, viing ITS va ITS2 noi len nhu DNA chi
' Truong Dai hpc Nguyen Tat Thanh
^ Vien Nghien ciru Khoa hoc Tay Nguyen ' Vien Di truySn Nong nghiep
"" Trucmg Dai hpc Su pham TP. HCM
thi giiip chi ra quan ht chung loai ciia Cymbidium chau A, dac biet vimg ITS2 co the dupe sir dung nhu mot ma vach DNA o miic dp cao cho cac loai lan (Sharma era/, 2012).
Tren ddi tuong Paphlopedllum, m6t nghien cuu phat sinh chung loai o miic dp phan tu da dupe thuc hien dua tren viec thu thap cac dii Ueu trinh tu DNA tir cac vi tri trong nhan (viing ITS) va trong plastid (mpt phan gen maQ^. ycfl, cac viing non-coding psaN-yc&ey3> va fi^;?F-ndhJ (Chochai et af 2012).
Trong nuoc, Khuat Hiiu Trung va cac cpng su cung da su dung trinh tir vung ITS de phan biet 16 loai va 2 thii duoi loai cua chi Papbiopedilum Viet Nam (Khuat Hiiu Trung et al., 2008). Nghien ciiu ket luan rang cac viing co dp bie'n thien cao o viing ITS rat hiiu ich cho viec phan tich phat sinh loai.
Nhu vay c6 hhi^u vimg trinh tir duoc su dung tren cac ddi tupng khac nhau cua hoa lan. Trong nghien ciiu nay, da sii dung 6 vimg trinh tu ITS, maK, tmL, rpd&, ipcCl, tivH-psbA de phan tich dac diem trinh tuva nhan dien loai lan Hai dac hiru Viet Nam cung nhu mot so cac loai lan Hai Viet Nam Ian can khac.
N O N G NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 1 /2020
KHOA HOC CONG N G H E
2 . VAT UEU VA PHUONG PHAP 2.1.V|itMu
Hai mucri mau la tir cay trucmg thanh da co hoa, thupc 8 loai lan Hai dac hiiu Viet Nam gom P.
delenatii, P. hangianum, P. helenae, P.
tranlienianum, P. x dalatense, P. gratrixianum, P.
vietnamense, P. x hermannn dupe thu thap tir Vien Sinh hoc Tay Nguyen va Vien Di truyen Nong nghiep. Trir P. herrmannii va P. vietnamense la 2 loai hiem, gan nhu tuyfit chung ngoai thien nhiSn nSn chi thu duoc 1 mau, cac loai con Iai d^u thu tir 3 rnm. tro len de so sanh.
BSng 1. Cic mdi sii dung trong nghifin cihi
2.2. T ^ h DNA t6ng s6, khu&h d?ii vjl giai trinh tii ckc vung trinh ti^ chi thi nh§n di^n
DNA tong so duoc tach bang Kit ISOLATE U Genomic DNA Kit, Cong ty BIOLINE. Hai muoi mau nghidn ciiu dupe su dung de khuech dai 6 viing trinh tu khao sat la FTS, maK, tmL. ipoB, ipoCl, tnM- psbk. Cac moi khuech dai trinh tir dupe neu a bang 1. Nhiet dp Tm de khuech dai cae viing ITS, /na/K, tinL, rpoB. rpoCl, tmU-psbA \ki luot la 58''C, 55"C, ea^c, ss-'c, ss-'c, 53x.
T6n viing trinh tir
ITS matK.
b-nL tpoB ipoCl tmU-psbA.
Ten moi ITl-F IT2-R F56-mo R1326-mo
tmh-¥
tmh-R 2F 4R I.IF 1.3R psbA3'f
Trinh tu moi AGTCGTAACAAGGTITC GTAAGTTTCTTCTCCTCC CCTATCCATCTGGAAATCTTAG
GTFCTAGCACAAGAAAGTCG GGTAGAGCTACGACTTGATT CGGTATTGACATGTAAAATGGGACT
ATGCAACGTCAAGCAGTTCC GATCCCAGCATCACAATTCC GTGGATACACTTCTTGATAATGG TGAGAAAACATAAGTAAAGGGC CGCGCATGGTGGTTCACAATCC
Chieu dai siin pham khuech dai
900 1100 600 600 600 900
Trich din (Tsai, 2011) Thiet k^ moi (Dang Van Khai
etal., 2017)
(Group 2009)
2.3. Phan tich du hfeu trmh hi
Phan mem Finch TV (Geospiza, 2004), dirpe sir dung d^ kiem tra dp tin cay cac peak tin hieu. Dft lieu trinh tu tho duoc kiem tra do tin cay va hieu chinh bang each doi chieu trinh tu chieu xudi vdi chieu nguoc va kiem tra peak tin hieu tai cac vi tri da hinh nucleotide trfin phan mem Seavievi' 4.0 (Gouy et al, 2009) va so sanh vdi trinh tu tir co sd dii lieu GenBank bang cong cu BLAST.
Phan mem MEGA (Kumar etal., 2008) duoc su dung cho viec tinh toan cac thdng sd ddt bien thay the nucleotide g6m Variation, Pasimony, Smgleton theo md hinh Kimura 2, thuat toan Maximum Likelihood vdi chi sd boostrap lap lai 1000 Mn. Cac thdng sd Monoindel, Insertion, Deletion dugc ghi nhan thu edng.
3. m QUA VA THAO LOAN
3.1. K^t qua thu mlu cac loM Ian Hai dac hmi Viet Nam
Nghidn ciiu da thu thap dupe 23 mSu lan Hai, thudc 8 Ioai dac hiiu gdm 6 loai nguyen thuy P.
delenatii, P. gratrixianum, P. hangiamnum, P.
helenae, P. tranlienianum, P. vietnamense va 2 loai lai P. dalatenseva P. xhermannii.
3.2. K^t qua tich chi^t DNA tdng s6, k h u ^ h d^i vii giai trinh tii cic vting trinh ttr chi thi nham di$n
DNA tdng sd cua 23 mlu lan duoc sii dung de khuech dai 6 vung trinh tu ciia lan Hai la ITS. maS^, OnL, rpoQ. rpoCl, tinU-psbH. Trong dd 5 vimg trinh tu ITS, ma/K, iJ-nL, ipoB. rpoCl eho ket qua khuech dai PCR thanh cdng tren tat ca cac mlu nghien ciiu ddng thdi hieu suat giai trinh tu cung dat 100%. Cap moi mam- 2.1F/5R d^ xuat boi CBOL (2009) hoi khd khu^eh dai hon cae viing trinh tu khac (Chase et al., 2007, HoUingsworth etal, 2009). Tuy nhien cap moi 56F-mo/1326R-mo do nhdm nghien eiiu thiet ke (Dang Van Khai etal, 2017) thi hieu qua khuech d;)i cao hon va it phai lap lai phan iing PCR. Cap indi ITl/rr2 duoc diing trong eac nghien ciiu trude day (Tsai et al, 2003, Gigot er al., 2007) eung cho hieu
KHOA HOC CONG NGHE qua khuech dai cao vimg ITS tren doi tirong Ian Hai
Papbiopedilum. Ba vimg trinh tu tniL, rpoB va /poCl d^ dang duoc khudch dai thanh cdng nh^t ngay phan ling dau tien.
Rieng viing tnii-psbA, cd 4/23 mlu khudch dai khdng thanh cdng. Hieu suat khuech dai chi dat 82,61%, day cung la hieu suat dat dupe sau rat nhieu lln lap lai phan ling PCR. Ngoai ra khoang each vach san pham chi dai khoang 600 bp thay vi 900 bp nhu du kien. Ket qua giai trinh tu vung tmU-psbA deu cho ket qua tin hieu trinh tu hi nhilu, dong thoi chi^u F chi nhan dupe chieu dai trinh tu khoang 200 bp thay vi 600-900 bp nhu du kien. Viing triM-psbA dupe de xuat lam trinh tu ma vach trdn lan Dendrobium (Yao et al., 2009) nhimg khdng thich hpp cho doi tupng lan Hai Papbiopedilum. Ket qua nay ciing hoan toan phii hop vdi mdt so cdng bd trudc day (Gigot etal., 2007, Dong etal, 2015). Vi th^, vung tinR-psbA da dirge loai bd khdi eac phan tich tiep theo.
Trinh hr cua timg mlu dirpc so vdi ngan hang dir heu gen GenBank b ^ g cdng cu BLAST. Ket qua eho thiy 23/23 mlu deu thudc nhdm lan Hai Papbiopedilum va dilng vdi cac viing trinh tu khuech dai, vdi dp tirong dong dat 95 - 100%. Trinh tir cac mlu sau khi kiem tra tinh chinh xac da dupe dang Id ma s6 tren ngan hang GenBank.
3.3. Dac dilm trinh ttr cua cic viing gen nghifen cihi
Khi phan tich bidn di di truydn eac mlu lan Hai trong nghien eiiu, viing ITS ed ti le Parsimon, Singleton, Insertion va Deletion cao nhat, ke den la maSi (Bang 2). Ti Ie thap nhat la 2 viing ipdB va rpcCl. Tinh da hinh di truydn eao eua hai viing FTS va maSi cung da dupe bao cao trude day
(Chattopadyay (Chattopadhyay et al, 2017. Parveen et al, 2017). Ngupe lai cac trinh tu tmL, ipdB va /pcClkhabaotdn.
Locus
ITS matK
trnL rpoB rpoCl
Bing 2. Da dgng di truy Chieu
dai trinh tu
661-703 1089- 1131 415-428
485 462
Chieu daa phan
tich (L) 725 1132 466 3213 1345
^n cic vuni Bi^n di thay the
Variable site So lugng vi
tri Parsimony
(P) 64 37 17 7 5
So luong vitri Singleton
(S) 108 46 9 14 13
; trinh tu ciia 8 loM lan Hai die hflu Viet Nam Bien di them/mat
Indel So luong
doan chen (Insertion)
4 0 0 0 0
So Iugng doan mat (Deletion)
1 0 0 0 0
So luong chen/mat 1
nu (Monoindel)
11 0 0 0 0
Tdng Indel 16
0 0 0 0
Tdng sd loai phan dinh dugc 3 5 4 3 3
Tyle loai phan dinh
(%)
37,5 62,5 50 37,5 37,5 Bang 3. Khoang d c h di truydn trong ding loli (intra- specific distance) v l khoang each di truyM giiia cic
loli (mter-speciflc distance) ciia mlu Ian Hii Viing trinh
Ur ITS matK
tmL rpoB rpoCl
Khoang each di truyen cimg loai (intra-specihcdistance)
0 0 0 0 0
Khoang each di truydn khac Ioai (inter-specificdistance)
0,020 0,000 - 0,076
0,008 0,000-0,029
0,009 0,000-0,031
0.006 0,000-0,022
0,003 0,000-0,011
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 1 /2020
Dac diem da hinh trinh tu (nucleotide polymorphism) ctia cac mau nghien ciiu cung dupe phan tich va so sanh (Hinh 1-3). Ngoai tni P.
dalatnese, cac mlu trong ciing loli deu cd dac diem trinh tir tuong ddng 100% ddng thoi the hien khoang
KHOA HQC CbNG NGHl
khoang each di truyen cao nhat (0,02) va each biet dang ke so voi 4 vimg trinh tu cdn lai.
Su tuong ddng trong loai the hien do bao ton trinh tu cao qua cac th^ he loai va d cae vimg dia ly khac nhau tren dat nude Viet Nam. P. dalatense iig i.\)\jm uuiig Liiui liic iiicii miwtuig KJiae iinau iren aat nude Viet Nam. P. dalatense cleh di tmyen ndi loai 110 (Bang 3). Khoang each di mang nhieu diem bien di trong loai. dieu nay la do P.
truydn toi thieu giiia eac loai khac nhau d ca 5 vimg dalatense Aver. 2002 la Ioai lai tu nhien giura P.
trinh tu deu bang 0,000 va khoang each tdi da bang callosum va P. villosum nen din den dac diem di 0,076 d vimg trinh tu ITS. ITS cOng cho trung binh truyen cd bien ddng hon so vdi cac loai nguyen thuy.
f4S'?,?^^^is^tS^^.?^.?ss^seg^f^ig$f$ssr?ss'f?^g.^j?si'gs$is^sf^sssgf.fSs^sssfs^s&s
Hinh 1. Da hinh nucleotide vung trinh h? FTS (sd thii tii ben trfen: vi tri nucleotide trong day trinh tu phan tich;
d^u chim: nucleotide gi6ng vdi nucleotide cung vi tri cua trinh tu diu tidn DEL'^2; diu gach ngang: vi tri khoang trdng gap)
' Hmh 3. Da hinh nucleotide vtrng trinh tu tn^' ~ -^T^
Hinh 2. Da hinh nucleotide viing trinh tu matK vl rpoCl
KHOA HOC CdNG NGHE 3.4. Nh$n difen loli dua vio cSy phat sinh (tree-
based method)
Muc dich eua nghien ciiu nay la nhan dien cac Ioai lan Hai Viet Nam ndn viec phan tich chi tiet kha nang phan dinh Ioai cua timg trinh tu ma vach duoc thuc hi&n trdn eae trinh tu Vidt Nam. Cae ma vach duoc khao sat bing timg vimg trinh tu rieng le va vung ghep 2 trinh tu. Loai duoc xem la phan dinh dupe khi eac trinh tu eua cimg loli dupe xdp vol nhau vao cung mdt nhanh don nganh tren cay phat sinh va tach biet vdi cac nhanh loli cdn lai.
Ddi vdi 5 viing trinh tu ridng le, madC cho kha nang phan dinh tdt nhat (5/8 loai) dat 62,5% (Hinh 4), cac trinh tu con lai ITS, tinL, rpoB va ipcCl deu dat 3/8. Viec ghep 2 trinh tu cho phep nhan di6n
duoc 6/8 loll dua vao vimg ma vach maK+YLS (Bang 4). Ket qua nay cQng phu hop voi d^ xuat cua Chochai va cpng sir (2012) v6 viec ket hpp mdt vimg trinh tu tir nhan (ITS) va mdt viing trinh tu tir plastid de tang hieu qua nhan dien (Chochai et al, 2012).
Ket qua danh gia cao ITS va maG^ ve kha nang phan dinh loll da dupe nhlc den trong nhieu nghien ciiu trudc day va k^ ca nhirng nghien ciru mdi day nhat (Chattopadhyay et al, 2017, Ghorbani et al., 2017, Parveen etal, 2017). Viec maO^ ket hpp vdi cac trinh tu khic (maK+dnL, maK+rpoB. maK+rpeCl) cho kha nang phSn dinhn loai (5/8) cao hon so voi khi ITS ket hpp vdi cac trinh tu (TTS+tivL. YYS+rpoB, TlS+rpoCl) (Bang 4). Viec ghep thu 3 trinh tir trd I6n d^u khong lam tang thdm kha nang phan dinh Ioai.
Bang 4. Kha nang ph§n dinh loli cua cic vijing trinh tu ma vach
natK trnL rpoB rpoCI malK+rrS maK+ttht'KmaK*rpoB maIKtrpoC1;IITS+lrnL rrS*rpoB fTSMpoCI
5 37 5 37 5 37 5
Kha nang phan dinh loai (%)
IHII lllliii
Ma vgch
Hinh 4. Bilu do Idia nang phSn dinh loai cua cic viing trinh tu ma vach
Ba loli P. delenatii, P. bangianum va P.
vietnamense deu duoe phan tach nhanh rd rang d tit ea eac cay phat sinh ghep va neng le. Day la 3 loll cimg thudc dudi chi Parvisepalum kha dac tnmg trong he th6ng phan loai hinh thai ciia chi Papbiopedilum. Trong khi do cac loai P. helenae, P.
tranlienianum, P. gratrixianum deu duoc xep cimg to (section) Papbiopedilum eua dudi ehi Papbiopedilum
nen eo quan he rat gan nhau v^ hinh thai lln di truydn. P. dalatensevk P. herrmanniila hai loai Iai tu nhien dupe khao sat trong nghien ciiu. P. berrmannn 11 Ioai lai hi bo me la P. birsubssimum va P. belenae cflng tiiudc cimg to nen vide ph^n nhanh cac Ioai nay nam eiing nhau la hoan toan cd co sd. P. berrmannii vl P. tranlienianum vin chua phan dinh duoc trong nghien ciiu. Day deu la loli dac hiiu cua vung niii phia Bac Viet Nam. Vide nhan dien 2 loli nly can cd thdm thdng tm phan bi hoac cd the phdi hpp vdi thdng tin hinh thai. Do P. berrmannii hien nay hau nhu da tuyet chung ngoai thien nhidn, nen chi thu duoc 1 mlu, do dd ehua cd phan tich so sanh di truyen cl the ciing loai. Cdn Ioai Iai P. dalatense thi the hidn su da dang di truyen rd trong ciing loai khi eac ca the trong loai thudng tach nhanh rieng so vdi 2 mlu chi em ciia nd (Hinh 5). Trong khi dac diem viing ITS eiia 3 mlu nay giong nhau, viee ghep maK.
vdi ITS eho kha nang tach 3 mlu Ioai P. dalatense thanh mdt nhanh ddc lap giiip nhan dien dupe loai nay trong 8 Ioai nghien ciiu. Cac trinh tu ciia loli P.
dalatense dupe dang ki tren ngan hang GenBank
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 1/2020
KHOA HOC CONG N G H £
c u n g la n h i m g t r i n h t u d a u tien va d u y n h a t c h o d e n n a y c u a Ioai nay, k h a n g d i n h s u d a c h i m loai c u a Viftt N a m .
P gratnxQnumGRA-ISO P gratnxianumGR.V182 P. gralnxianum GRA-1^6 P. gralraoanum GRA-145 p P dalaIenseOAL-143
—-1 P dalalense DAL-t38 ' p dalatense OAL-139 . « . , P » l . n . . H E L . 7 0
- i f '-e[ecaeHEL.71 ' p helenae HE L-69 P hefTmannnHeR-177 P iranlienianum TRA.63 P iranhenianum TRA-BS P iranlienianum TRA-SJ
• P hangianum HAN-17
^^ _ _ J p hangianum HAN-lB 1— ' P hangianum hlAN-IB 1 P detenalii DEL-2
P detenaui DEL-46 delenatii DEL-47 P delenatii DEL-187
h delenatu DEL-188
H i n h 5. Cay p h a t s i n h loai ITS+matK 4. KFT LUAN
T r o n g n g h i e n ciiu d a x a c d i n h d a c d i e m c u a 5 v i m g trinh t u p h a n dinh loai ITS, mam. tmL, / p o B , rpcCL T h d n g tin t r i n h t u c a c loai Ian Hai Viet N a m d a d u p e d a n g k i m a s d trinh tii t r e n n g a n h a n g G e n B a n k , g d p p h a n v a o b d s u u t a p t h d n g tin p h a n tir t h e gidi, d o n g thdi d a n g ki d a c t n m g t r i n h tir c u a loai Vidt N a m . C a c v u n g tniR-psbP^ ipoB, ipcCl k h d n g phil h p p d e I a m t r i n h t u m a vach. Vide k d t h o p 2 viing trinh t u maK va I T S d a n h a n dien t h a n h c d n g 6 / 8 l o l l lan Hai d a c h u u Viet N a m . K^t q u a n a y la c o s o d e m d r d n g vide n h a n dien t r e n q u l n t h e lan H a i Viet N a m . Kdt q u a n g h i e n ciiu n e n d u p e a p d u n g v a o c d n g t i e q u a n li v a b a o tdn n g u o n g e n t h u e vat cd g i a tri e u a Viet N a m , d d n g t h d i g o p p h i n k h i n g d i n h b a n q u y e n d a c hiiu Vidt N a m c u a c a e loai q u y h i e m d n u d e t a .
TAI UEU THAM KHAO
1. A s a h i n a H , Shinozaki J, M a s u d a K, M o r i m i t s u Y a n d S a t a k e M (2010). Identification of m e d i c i n a l Dendrobium s p e c i e s b y p h y l o g e n e U c a n a l y s e s u s i n g m a t K a n d r b c L s e q u e n c e s , fournal of Natiiral M e t / / c m e s 64(2): 133-138.
2. C h a s e M W , C o w a n RS, HoUingsworth P M , van d e n B e r g C, M a d r i n a n S, P e t e r s e n G, S e b e r g 0 , j 0 r g s e n s e n T , C a m e r o n K M , C a r i n e M , P e d e r s e n N , H e d d e r s o n T A J , C o n r a d F , Salazar G A , R i c h a r d s o n J E , HoUingsworth M L, B a r r a c l o u g h T G, Kelly L a n d Wilkinson M (2007). A p r o p o s a l for a
s t a n d a r d i s e d protocol t o b a r c o d e all land pi"- rAYOA^56(2): 295-299.
3. C h a t t o p a d h y a y P , Banerjee G a n d Bai' (2017). Distinguishing O r c h i d S p e c i e s b \ .'"NA B a r c o d i n g : I n c r e a s m g t h e Resolution of Population S t u d i e s in P l a n t Biology. Omics2\(\2): 711-720.
4. C h o c h a i A. Leitch I J, higrouille M J a n d F a y M F (2012). M o l e c u l a r p h y l o g e n e t i c s of P a p h i o p e d i l t u n (Cypripedioideae; O r e h i d a c e a e ) b a s e d o n n u c l e a r ribosomal I T S a n d plastid s e q u e n c e s . Botanical Journal of the Unnean Society 170(2); 176^196.
5. D a n g V a n Khai, N g u y d n Tlii N h a v a V u T h i H u y ^ n T r a n g (2017). S c r e e n i n g , d e s i g n i n g , pilot evaluation a n d application of s o m e potential p r i m e r s for m o l e c u l a r discrimination of Papbiopedilum s p e c i e s in V i e t n a m . T a p c h i N d n g n g h i e p v a P h a t t r i e n n o n g t h d n : 113-118.
6. D o n g W , Xu C, U C, J i a h u i S, Zuo Y, S h i S, C h e n g T , G u o J a n d Z h o u S (2015). ycft, t h e m o s t p r o m i s i n g plastid D N A b a r c o d e of land plants.
Scientific reports^: 8348.
7. Geospiza I (2004). F i n c h T V version 1.4.0:
D N A s e q u e n c e c h r o m a t o g r a m t r a c e viewer.
8. G h o r b a n i A, G r a v e n d e e l B , Selliah S, Z a r r e S a n d d e B o e r H (2017). D N A b a r c o d i n g of t u b e r o u s O r c h i d o i d e a e : a r e s o u r c e for identiEieation of o r c h i d s u s e d in Salop. Molecular Ecology Resources 17(2):
342-352.
9. G i g o t G, Van Alphen-Stahl J , B o g a r i n D, W a r n e r J, C h a s e M W a n d Savolainen V (2007).
F i n d i n g a suitable D N A b a r c o d e for M e s o a m e r i e a n o r c h i d s . Lankesteriana Intemational Joumal on Orcbidology7(X-2): 200-203.
10. G o u y M , G u i n d o n S a n d G a s c u e l O (2009).
SeaView V e r s i o n 4: A Multiplatform Graphical U s e r Interface for S e q u e n c e A l i g n m e n t a n d P h y l o g e n e t i c T r e e Building. Molecular Biology and Evolution 27(2): 221-224.
11. G r o u p C P W (2009). A D N A b a r c o d e for land plants. Proceedings of the National Academy of Sciences of die United States of Amenca 106(31):
12794-12797.
12. HoUingsworth P M , F o r r e s t L L, S p o u g e J L, Hajibabaei M , R a t n a s i n g h a m S, van d e r B a n k M , C h a s e M W , Cowan R S, E r i e k s o n D L, F a z e k a s A J , G r a h a m S W, J a m e s K E , K i m K-J, Ki - '.vj.
S c h n e i d e r H, v a n A l p h e n S t a h l J, B a r r e t t SC^
d e n B e r g C, Bogarin D , B u r g e s s KS, C a m e r o n : .i,
N O N G NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG l/20„
KHOA HOC CONG NGHE
CanneM,Chac6nJ,ClarkA,ClarksonJJ,ConradF, loci (riicL, rpoB, rpoCl, matK, and ITS) for DNA Devey D S, Ford C S, Hedderson T A J , barcodingoflndian orchids. Ge/io/ne60(8): 66&^71.
HoUingsworth M L, Husband B C, KeUy L J, 16. Sharma S K, Kumaria S, Tandon P and Kesanakurti P R, Kim J S, Kim Y-D. Lahaye R, Lee H- Satyawada R R (2012). Comparative L, Long D G, Madrinan S, Maurin O, Meusnier I, karyomorphological study of some Indian Newmaster S G, Park C-W, Percy D M, Petersen G, Cymbidium Swartz, 1799 (Cymbidieae, RichardsonJE, SalazarGA,SavolainenV,SebergO, Orehidaceae). Comparative cytogenetics 6(4): 453- WiDdnson M J, Yi D-K and Uttle DP (2009). A DNA 465.
barcode for land plante. Proceedings of the National 17. Tsai C C (2011). Molecular Phylogeny and Academy of Sciences V^&Qiy. 12794-12797. Biogeography of Phalaenopsis Species. Orchid
13. Khuat Hiiu Trung, Tran Dang Khanh, Le Biotechnology IL 1-24.
Huy Ham, Tran Duy Duong vl Nguydn Trpng Khoa 18. Tsai C C, Huang S C, Huang P L and Chou C (2008). Evahiation of genetic diversity H (2003). Phylogeny of the genus Phalaenopsis Paphiopediltun groups in morphology; analysis of (Orehidaceae) with emphasis on the subgenus specific characteristic of indigenous gene resource of Phalaenopsis based on the sequences of the internal P^hiopedilum in Vietnam. Hanoi, Vietnam, Ministiy transcribed spacers 1 and 2 of rDNA.
of Science and Technology. 19. Yao H, Song J Y, Ma X Y, Uu C, U Y, Xu H X, 14. Kumar S, Nei M, Dudley J and Tamura K Han J P, Duan L S and Chen S L (2009).
MEGA: a biologist-centric software for Identification of Dendrobium species by a candidate evolutionary analysis of DNA and protein sequences. DNA barcode sequence: the chloroplast psbA-tmH Bn'eSngs in Bioinformati'cs9(i): 299-306. intergenic region. Planta Medica75(6): 667-669.
15. Parveen I, Singh H K, Malik S, Raghuvanshi S and Babbar S B (2017). Evaluating five different
IDENTIFICATION OF ENDEMIC P^hiopedUum SPECIES IN VIETNAM Vu Tlu Huyen Trang^, Vu Quoc Luan^, Le TTii Ngoc DiepS Nguyen TTianh DiemS Nguyen Tiianh Cong^, Luu Phuong NamS Nguyen Huu Thuan Anh', Tran Duy Duong^, Tran HopS Tran Hoang Dung^
^Nguyen Tat Thanh University
^TayNguyen ScientiSc Research Institute Central
^ Vietnam Agricultural Genetics Institute
^Education Umversity - HCMC Summary
Papbiopedilum is a rare and endangered orchid group. Vietnam possesses diverse species of this genus and some of them are valuable endemic taxa. Therefore, conservation measures are needed. Most illegal trading are apphed on immature, flowerless plants. Hence a fast and accurate identification method is needed for effecUve conservation. DNA barcoding is a new method, which is mcreasmgly popular today. In this study, to identify Paphiopedilum species, we investigated 6 sequence regions ITS, ma&i. tmL, rpd&.
rpoCl, tTTiii- on 23 samples of 8 Paphiopedilum species. The amplification of 5 regions ITS, inad^ trnL, rpoB, rpoCl were 100% successful. The sequences in this study were submitted and contnbuted to GenBank. The results of this study proposed the combination of ITS and matK sequences as the highest potential for identifying 6 Paphiopedilum species, including Paphiopedilum delenatii. P. hangianum, P.
helenae, P. x dalatense, P. gratrixianum, P. vietnamense. This barcode sequence can be applied to identify Vietnam Paphiopedilum population serving for management and conservation of this valuable orchid group.
Keywords: Paphiopedilum, endemic species, molecular identi£cation, ITS, matK, tmL.
Ngudi phan bidn: PGS.TS. Nguyin Thi Kim Ly Ngly nhan bii: 11/11/2019
Ngly thdng qua phin bidn: 11/12/2019 Ngly duy^t dang: 18/12/2019
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 1/2020 9
