Tap chi KHLN 3/2014 (3382 - 3389)
& Vien KHLNVN - VAFS
ISSN 1859-0373 Bang tai tai www vafs.gov.vn
DANH GIA DA DANG Dl TRUYEN CAC X U A T XU' CAY LAI {Aleurites moluccana (L.) Willd) BANG CHJ THI. PHAN TCT RAPD
Trdn Due Vugng', Biii Ngoc Quang', Luffng Van Tien^ Hoang Van ThSng^, Trdn H6 Quang ' Vien Nghien cim Gidng vd Cong nghe Sinh hoc Ldm nghiep
^ Vien Khoa hoc Ldm nghiep Viet Nam
^ Vien Nghien cuu Ldm sinh
TOM TAT
Da dang di truyin cua 31 cay Lai {Aleurites moluccana (L.) Willd) thugc nam xudt xu khac nhau (Bic Kan, Lang Son, Thanh Hoa, Nghe An va Gia Lai) da dupc danh gia bang bay chi thj phan tir RAPD da hinh (0PN16, OPH08, OPR08, OPALS, 0PAK14, 0PA4, OPAB5). Kit qua phan tich cho thdy cac chi thi RAPD nay c6 960 bang, kich thaac cdc phan doan ADN Tif khda: Cdy Lai, da nSm trong khoang tCr 100 - 950 bp vcri ty le bang RAPD da hinh dao dpng dang dl truyen. RAPD. j,^ 54^34 . 64,83%, So d6 m6i quan he hinh cay tir 31 cSy theo phuong phap UPGMA cho thdy hdu hit cac cay cua ciing mot xudt xu nSm trong cimg mpt nhom va dupc chia thanh 2 nhom lan vai he s6 tuang ding di truyin la 0,8. Nhom I gim 16 cay thupc xudt xii Thanh Hoa, Nghe An va Gia Lai CO he s6 sai khac v6i cac mdu khac la 0,16 (0,84 - 1,00) va du'ac chia thanh 3 nhom phu. Nhom II gom 15 cay thupc cac xuat xii Bdc Kan va Lang San va duoc chia thanh 2 nhom phu voi he so tucmg dong di truyen tir0,915-0,985.
Study genetic diversity oi Aleurites moluccana (L.) Willd's provenances by RAPD markers
Genetic diversity of 31 Aleurites moluccana trees from five provenances (Bac Kan, Lang Son, Thanh Hoa, Nghe An and Gia Lai) were evaluated by seven polymorphic RADP markers (0PN16, OPH08, OPR08, OPALS, 0PAK14, 0PA4, 0PAB5). Seven RAPD markers amplified 31 individual Keywords: Aleurites trees and produced 960 bands with size ranges trom 100 bp to 950 bp. The moluccana, genetic polymorphic level ranged from 54.34% to 64,83%, Dendrogram obtained diversity, RAPD from 31 trees with UPGMA method showed 2 main clusters with similarity coefficient of 0.8. Cluster I comprised 16 individuals from Thanh Hoa, Nghe An and Gia Lai and having similarity coefficient trom 0,84 to 1.00 and divided into three sub - clusters. Cluster 11 comprised 15 trees from Bac Kan and Lang Son provinces and contained 2 sub - clusters with similarity coefficient from 0.915 - 0.985.
Tr^n Bifc Vu-yng et al.. 2014(3) Tap chi KHLN 2014
I. DAT VAN m
Cdy Lai (Aleurites moluccana (L.) Willd) la loai thuc vat thdn g6 thuoc ho Thau dau (Euphorbiaceae), 6 Viet Nam loai cay nay co phan bo tir cac tinh phia Bac nhu Cao Bang, Lao Cai, Lang Son, Bac Giang, Thai Nguyen...
den cac tinh mien Trung nhu Thanh Hoa, Nghe An, Quang Nam va cac tinh Tay Nguyen, Gia Lai, Dik Ldk... Ddy la loai cay ua sang, sinh trudng nhanh, ua ddt tit, tdng dat sau tren cac nuong rdy cii hoac ddt xung tich da voi. Lai la loai cay co kha nang tai sinh hat va choi deu rat tot.
De tai cap Bo "Nghien ctru chon giong va ky thudt trong cdy Lai (Aleurites moluccana) d Tay Nguyen, Bac Trung Bo va Dong Bac theo hudng liy qua" (2010 - 2014) do TSKH.
Luong Van Tien, Vien Khoa hoc Lam nghiep Viet Nam chii tri dugc thuc hien, vdi muc dich xac djnh dugc cac bien phap gdy trong phil hgp va xac dinh cac gidng Lai co nang suat qua, ham lugng va chdt lugng dau cao, dong thdi de tai ciing nghien ctru da dang di truyen cua 31 cay Lai (Aleurites moluccana (L.) Willd) thuoc nam xudt xu khac nhau (Bac Kan, Lang SOTI, Thanh Hoa, Nghe An va Gia Lai) dugc danh gia bang bay chi thi phan Xii RAPD da hinh.
Dll mdi dugc di vao nghien ctru, khai thac va sir dung d Viet Nam nhimg co the thay rang Lai la cdy c6 gia tri sir dung cao dac biet la tilm nang ve khai thac ddu Diesel sinh hoc.
Day la su thay the nguon nguyen lieu truyen thong gdy 6 nhiem moi trudng va dang ngay mpt can kiet (Luong Van Tien et al, 2012). Do
vay, viec lira chpn nguon vat lieu giong ban dau la het sue quan trpng va la nhiem vu cap thiet trong cong tac bao ton, khai thac va phat trien nguon gen cay ban dia co gia tri kinh te.
Ngay nay, ngoai muc dich chpn giong theo tinh trang muc tieu, viec duy tri mgt mirc dp on dinh ve da dang di truyen trong cac quan the chpn tao giong trong cac the he de dam bao tinh on dinh va Hnh hoat cua nguon gen luon dugc quan tam. Viec danh gia da dang va quan he di tniyen cac quan the chgn tao giong la mpt cong viec khong the thieu trong chuong trinh chgn giong cdy lam nghiep.
Chi thi phan tii RAPD (Random Amplified Polymorphic DNA - Da hinh ADN nhdn ban ngdu nhien) la chi thi dua tren nen PCR vdi mgt moi don co trinh tu ngdu nhien gom khoang tir 5^12 nucleotide (Williams el al, 1990). Chi phi cho viec sir dung m6i RAPD nay khong cao nen thudng dugc sir dung de danh gia da dang di truyen cho nhieu loai cay rimg ban dia nhu Coc rao (Jatropha curcas) (Basha va Sujatha, 2009), Md Hai Nam (Manglietia hainanensis Dandy) (Nguyen Hoang NghTa et al, 2009), Sd (Camellia sp.) (Nguyen Quang Khai va Khuat Hiru Trung, 2007), Go do (Afzelia xylocarpa Kurz) (Nguyen Hoang NghTa et al, 2007).
II. VAT LIEU VA PHirONG PHAP NGHIEN ClTU
2.1. Vat lieu
Trong nghien cun nay chung toi da sir dung mdu la cita 31 ca the cay Lai tir nam xuat xu khac nhau dugc trinh bay trong bang 1.
Bang 1. Cac xuat xu cdy Lai dugc sii dung trong nghien cuu
STT 1 2 3 4 5
Oja diem Idy m l u Bic Kan
Lang San Thanh Hoa Nghe An Gia Lai
S6 liFcng cay 8 7 6 3 7
Ky h i f u BB: 1 . 2 . 5 . 7 , 8 , 9 , 11,12 CL:1,2. BS 5 , 6 . 7 , 9 , 12 QH:6, 7, 8, 9. 11. 13 AS: 1.2, 3 PL: 1,5,7,8,9, 10, 11
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Tap chi KHLN 2014 Tran Bii-c Vyqnq ef al.. 2014(3)
Cac moi ngau nhien sir dung trong nghien cuu ia nhirng moi co miic dp da hinh cao va da dugc sii dung trong cac nghien cim trudc
day cho cac loai cdy ban dia. Bay moi ngdu nhien dugc sir dung trong nghien ciiu dugc trinh bay trong bang 2.
Bang 2. Danh sach va trinh tu bay moi ngau nhien dugc sir dung
STT 1 2 3 4 5 6 7
Ten moi 0PN16 OPH08 OPR08 OPALS 0PAK14 0PA4 0PAB5
Trinh ti/ (5' - 3') AAGCGACCTG GAAACACCCC CCCGTTGCCT GTCGCCCTCA CTGTCATGCC AATCGGGCTG CCCGAAAGGGA
2.2. PhiroTig phap nghien ciiii Thu vd bdo qudn mdu nghien ciru Mau de danh gia da dang di truyen la la cay Lai sach benh, khong qua non va khong qua gia (la banh te) dugc thu tai cac cdy d cac xuat xtj khac nhau. Mau la sau khi lay ve phong thi nghiem se dugc nghien thanh bpt min vdi ni to long va bao quan d nhiet dp - 80"C cho tdi khi sir dung.
Phuong phdp sinh hpc phdn tu
ADN tong so dugc tach chiet theo phuang phap CTAB thong thudng co cai tign (Gawel va Jarret, 1991). Ham lugng va chit lugng ADN dugc kiem tra bang phuong phap dien di tren gel agarose 1% va may do quang ph6 Nanodrop.
Phan ling PCR vdi cac moi ngau nhien dugc tien hanh vdi tdng the tich la 20pl/lmau g6m nhiing thanh phan trong bang 3.
Bang 3. Thanh phdn phan img PCR - RAPD
Thanh phin phan i>ng HjO
Primer (lOmM) PCR IVIaster Mix DNA (25 ng/pl) Tong th6 tich phan irng
Th^ tich sir dung (pi) 6 2 10 2 20
Chu trinh phan img PCR gim 94°C trong 3 phut, sau do la 40 chu trinh gom (94°C trong 1 phut, 36^C trong 30 giay, 72°C trong 1 phiit), 72°C trong 10 phut va duy tri a 12''C.
San pham PCR sau d6 dugc dien di tren gel agarose 2,5% va nhugm bing Ethidium Bromide, sau do quan sat duoi tia UV va dugc chup iinh bang may UV - Vis.
Phdn tich moi quan he di truyen
Cac phan doan DNA dugc ghi nhan dua tren su CO mat hay khong co mat ciia chiing 6 cac mau nghien cuu theo thang ADN chuin (DNA maker). Neu xuat hien bang ADN thi ky hieu la 1 con neu khong co thi ky hieu la 0. Cac si lieu nay sau do dugc xu ly bSng phin mim NTSYSpc 2.2 (Rohlf, 2008) di tinh ma tran hrong dong giiia cac doi mlu theo phuong phap UPGMA theo mirc do xa gan vi mat di tmyin va ve thanh sg di hinh cay hong TREE DISPLAY.
Ma tran tuong ding va khoang each di truyin dugc thiet lap dua vao cong thiic cua M. Nei va Li (1979).
Sij = 2N,/(N,-i-Nj)
Trong do: N,: so vach eiia giong i; Nj! si vach cua giong j ;
N,,: so vach triing nhau ciia hai giong I vaj; $„: he so ding dang.
Tran DiJc Vu-gng etat., 2014(3) Tap chi KHLN 2014
III. KET QUA VA THAO LUAN 3.1. Ket qua phan tich mirc do da hmh Trong nghien ciiu nay chiing toi da sii dung bay moi ngau nhien: 0PN16, 0PA4, OPH08, OPR08, 0PAB5; 0PAL8 va 0PAK14 de phdn tich miic dp khac nhau vh da dang di truyen ciia 31 mdu Lai. Ket qua
nhan dugc ca bay moi deu cho bieu hien da hinh kha ro rang, dieu nay kha thudn lgi cho viec phan tich tinh da dang ADN khi so sanh tat ca cac mau vdi nhau. Dien di san pham RAPD vdi bay moi ciia 31 mau Lai thu dugc tong cgng 960 bang, kich thudc cac phdn doan ADN nam trong khoang tir 0,1 dgn 0,95 kb.
Hmh 1. San pham PCR cua cac m i u Lai vdi mo! OP AK14 Ghi chu: So thii tu trong anh la so mau nghien ciru, M: DNA marker I KB
Cac phdn doan ADN dugc nhdn len bang cac moi RAPD dugc thong ke bang ma nhi phdn va ket qua dugc trinh bay d bang 4 dudi ddy.
Bang 4. Tong hgp cac bang ADN xuat hien va da hinh ciia 31 cay Lai khi phdn tich bdng chi thi RAPD vdi 7 moi ngdu nhien
X u i t xi> (ky h i f u m i u ) Bac Kan (BB) Lang San (BS, CL) Thanh Hoa (QH) Gia Lai (PL) Nghe An (AS) Tir cac xuit ^ir
Sd do^n da hinh 177 151 104 100 46 578
Tong so doan 273 245 176 184 82 960
Ty le doan da hinh chilm (%) 64,83 61,63 59,09 54,34 56,09 60,20
Bang tren cho thay, ty le bang da hinh ctia cac xuit xii dao dgng tir 54,34 - 64,83% vdi miic dao dgng la trong mpt khoang gia tri nhd (~ 10%) chiing to cac xudt xti co miic dp tucmg doi dong deu ve miic dp tuong ddng di truyen.
3.2. Ket qua phan tich moi quan he dl truygn Moi quan he di truyen ciia nam xudt xii cay Lai dugc tinh toan bang chuang trinh NT SYSpc 2.2 vdi thuat toan tinh ma trdn tuong dong giira cac doi mau theo phuang phap phdn nhom UPGMA theo miic dp xa gdn ve mat di truyen dugc thg hien d bang 5 va so do hinh cay dugc thg hien trong hinh 2 dudi day:
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Bang 5. Bang ma tran tucmg dong ciia 31 cay Lai
|,., |.-. | „ . |..u |.... |,r. |r.. |,r, | „ . |.» |,u. |,... |... |..: |.., |.., |... |... |...r |...= |... |.» |^^. \^-' \'--' \'=
SSZ 0.9J6.0 $Bt I
Tran BLPC Vu-p-ng ef al., 2014(3) Tap chi KHLN 2014
U EQ m D to m cS I
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Tap chi KHLN 2014 Trin eirc Vugng et a/., 2014(3)
So di mii quan he di truyen hinh cay cho thdy h4u hit cac ca the ciia cimg mgt xuat xir nam ciing mpt nhom vol nhau va 5 xuat xu co thi chia thanh 2 nhom lon co he so tuong dong di truyen la 0,8:
Nltom I: gim 16 cay thugc cac xuat xir Thanh Hoa, Nghe An, Gia Lai co he so sai khac voi cac mau khac la 0,16 (1 - 0,84) va dugc chia thanh cac nhom phu sau:
Nhomphij l.l: gom 7 cay ciing co xuat xir tir Gia Lai (PLl, PL5, PL7, PL9, PLU, PL8, PL 10) CO he so tuong dong di truyen cira cac cay tir 0,91 - 1,00.
Nhom phil 1.2: gom 4 cay thugc xuat xu Thanh Hoa (QH6, QH7, QH8, QHll) co he so tuong dong di truyen so vai cac nhom khac 14 0,91.
Nhom phil 1.3: gom 3 cay thugc xuat xir Nghe AmASl,AS2,AS3.
Rieng 2 cay QH9 va QH13 thugc xuit xir Thanh Hoa nhung lai tach biet han so vol cac cay con lai thugc ciing mgt xuAt xir.
Nltom 2: gom 15 cay con lai va dugc chia thanh 2 nhom phu.
Nhom phu 2.1: gim cac cay BB2, BBS, BB7, BB8, BB9, BBl 1, BBl2 CO he si sai khac voi cac mlu con lai la 0,05 (1 - 0,95), day la cac mhi diu thuoc quin the BJc Kan. Nhom nay lai dugc chia thanh 2 nhom phu.
Nhom phu 2.2: gim 8 cay CLl, CL2, BBl, BS5, BS6, BS7, BS9, BSl2 chu yiu thugc xuat xir Lang Son va co mirc do tuong ding khoang 0,91 va khong co mlu nao co mirc do tuong ding ion nhit la 1,00.
Ket qua moi quan he di truyin cua 31 cay Lai cho thay mirc do tuong ding thip nhit dugc ghi^ nhan la 0,714 giiia hai cay BBS (thugc xuat xir BSc Kan) va QH9 (thugc xuit xu Thanh Hoa). Diiu nay cung phii hgp vol kit
qui! ciia cay phan ioai (Hinh 2) khi hai cay Lai nam 6 hai nhom chinh c6 quan he xa nhau ve mat dia ly. Ly do mirc do tuong dong cua hai ciiy thiip nhu vay chu yeu la vi hai cay nay nim a hai quan the khac nhau va co khoang each dia ly kha lon (BSc Kan - Thanh Hda).
Dieu nay chimg to cac xuat xu Lai co nin tang di truyen cao, muc do da dang di truyen lon va CO mirc do phan bo kha rgng. Mat khac, viec nay ciing kha hiru ich vi nhu vay se lam phong phu them nguon gen, hieu qua hon trong viec chgn giong hay lai tao giong mod.
Ngugc lai, hai cay BBS (xuat xii Bic Kan) va BSl (xuat xir Lang Son) la hai cay thu tir hai quan the khac nhau (Bac Kan - Lang Son) c6 he so tuong dong cao nhat 0,95. Khong kho giai thich khi ma hai quan the nay co khoang each gan nhau hon. Theo tai lieu thu thap mau dugc can bg dieu tra cung cap thi hai cay nay dugc lay cimg mgt khu vyc. Vcri ket qua phan tich he so tucmg dong di truyin co thi khing dinh BBS va BSl co khoang each di truyin rit gan nhau.
Cac xuat xir cay Lai co khoang each dia ly kha xa nhau (Lang Son va Thanh Hoa) nhung lai CO he so tuong dong di truyin kha gSn nhau (0,84 - 1,0), diiu nay cung phii hgp vdi nghien cim truoc day cho cay ban dia nhu cay Mg Hai Nam (Manglietia hainanensis Dandy) (Nguyen Hoang Nghia et ai, 2009). Cac xuit xii Mo Hai Nam co khoang each kha xa vl mat dia ly (dao Hai Nam Trung Quic va Vudn Quic Gia Ba Vi - Viet Nam) nhung lai CO he s6 tuong dong di truyin tir 0,82 -1,00.
Nhu vay, trong nam xuat xil Lai nghien cini CO xuit xii Nghe An, Thanh Hoa, Gia Lai la cac xuat xir co tit ca cac mau nghien ciiu thugc cimg mgt nhom phan loai tuc la giiia cac mau nghien cim thugc xuit xii nay khong CO sir khac biet r<5 rang vi mat di truyen (he si) tuang dong di truyen trong quan the cao).
Trin BIFC Vugng ef al, 2014(3) Tap chi KHLN 2014
Dieu nay ciing phii hgp vdi cac nghien ciiu vdi mirc dao dpng nho (~ 10%), chiing to cac trudc ddy ve miic dp da dang di truyen cho xudt xii co su on dinh ve miic dp tuong dong cac loai cay ban dia nhu loai Po Mu (Fokienia di truygn.
hodginsii) dao dgng ttr 0,87 - 1,0 (Phi Hing ^^ ^ .^ ^ ^ ^ ^ ^ g , ^^^<, ^ ^ ^ . ^ ^^.^ ^.^ ^.^
Ha, va Nguyen Hoang NghTa, 2013). ^^^^ ^^^ ^ ^ , p^j„ „^ . ^ ^. ^. ^g „ ^ g IV KFT I TIAN dong di truygn dao dpng tir 0,80 - 1,00. Trong do 2 phan nhom ldn la (1) Thanh Hoa, Nghe Phan tich da dang di truyen 31 cay Lai thugc 5 ^^ ^-^ ^ai va (2) Bac Kan, Lang Son co xuat xii bang 7 chi thi RAPD cho thay ca 7 ^ ^ ^ ^g . ^ ^ ^ ^ . ^ ^ ^. ,^y>„ j ^ „ j . „ g ,j, chi th. nay thu dugc so bang la 960 biSng, kich ^^^^ ^ . ^ ^^g ^ ^ ^ j - , ^ ^ g j>„g g^-^ ^.^
thudc cac phdn doan ADN nam trong khoang ; . - u- i. •• i.- i,- '" -. .*• i - • . ^ • B & xuat xu phu hcrp vot phan bo ve mat dia ly cua tir 0,1 den 0,95 kb. Tyle bang RAPD da hinh , . , ,
; -^ • ^ cac xuat xu.
cua nam xuat xti dao dgng tir 54,34 - 64,83%
TAI LIEU THAM KHAO
1 Basha, S. D. and Sujatha, M., 2009. Genetic analysis of Jatropha species and interspecific hybrids ofJalropha cK/-ci3i'using nuclear and organelle specific markers. Euphytica 168, 197-214.
2 Gawel, N. J, and Jarret, R. L., 1991, A modified CTAB DNA extraction procedure for Musa and Ipomoea, Plant Molecular Biology Reporter 9, 262 - 266.
3. Luang Van Tien, Vu Hoang Phuong va Hoang VSn Thing, 2012, Ket qua nghien ciru hudc dau ve th^nh phSn hoa hgc ciia dau hat Lai (Aleurites molucana). Tap chi Khoa hpc Lam nghiep 2, 2273 - 2279.
4. Nguyin Hoang NghTa, Nguyin Diic Thanh, Tran Thiiy Linh, 2007. Ket qua phan tich da dang di truyen loai 05 do (Afzelia xylocarpa (Kurz) Craib.) bang chi thi phan ni RAPD Tap chi Nong nghiep va Phat trien nong thon 14,44-48,
5. Nguyin Hoang NghTa, Tran Thanh Trang, Do Tien Phat, Nguyen Van Phuong, Le Van Son va Chu Hoang Ha, 2009, Phan tich da dang di truyen he gen nhan ciia ioai Ma Hai Nam bang chi thj RAPD va cpSSR. Tap chi Khoa hpc Lam nghiep 2, 918 -925,
6. Nguyin Quang Khai va Khuat Htm Trung, 2007. Nghien ciru da dang di truyen mot s6 gi6ng So (Camellia sp.) Clia Viet Nam bang ky thuat RAPD. Tap chi Khoa hpc Lam nghiep 4, 452 - 459.
7. Phi Hong Hai va Nguyen Hoang NghTa, 2013. Bao cao ket qua cong tac bao ton va phat triln ngu6n gen cay lam nghiep tai Vien Khoa hpc Lam nghiep Viet Nam giai doan 2011 - 2013 va dmh huong cong tac den nam 2020, Tuyen tap bao cao Hpi nghj danh gia ket qua hoat dpng khoa hpc cong nghe ve quy gen (giai doEin 2011 - 2013). Nha xuit ban Khoa hpc va Ky thuat HaNpi - VietNam, 130- 148.
8. Rohlf, F. J., 2008. NTSYSpc: Numerical Taxonomy System, ver. 2.20. Exeter Publishing, Ltd.: Setauket, NY.
9. Williams, J. G. K., Kubelik, A. R., Livak, K, J., Rafalski, J. A. and Tingey, S, V., 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18, 6531 - 6535.
Ngudi tham dinh: TS. Bui Van Thang
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