Tgp chi Cong ngh? Sinh hpc 12(3): 515-522, 2014
BU"6C D A U N G H I E N C l T U , i T N G D U N G C H I T H I P H A N TU" I S S R T R O N G X A C D J N H C O N L A I C U A M O T S O T O H O P L A I X A C H I LILIUM T A I V I £ T N A M
Biii Thi Thu Hinmg''^, nh M a n h Tirdug', L£ VSn Som', Le T r a n B i n h ' , B a n g Vdn B 6 n g ^ Trjnh Khde Quang^
' Vi?n Cong nghi sinh hpc, Vien Hdn ldm Khoa hpc vd Cong nghi Viet Nam
^Hge vi?n Nong nghiep Viet Nam, Bg Nong nghi?p vd phdt trien nong thon Vi?n Nghiin cihi Rau qua, Viin Khoa hpc Nong nghiip Viet Nam
Ngdy nhgn bai:14.4.2014 Ngdy nhgn ddng:15.6.2014
Lily la mdt trong nhung logi hoa cd gid tn kinh te cao, mau sac dep, hoa thom, lau tan, dugc nhieu ngudi ua thich O nude ta hiSn nay, hoa lily dugc Uong chu yeu 6 Da Lat, Lam Ddng va mien BSc vdi sd lucmg vd quy mo ngay cang tang. Tuy nhien, Viet Nam cung nhu nhiiu nuac khac Uen the gidi chua san xuat dupc cii gidng boa lily dam bao ti€u chuan ma van phai nhgp cu giong tir Ha Lan vdi udc tinh gan 60 trieu cii gidng hoa lily cac logi cung iing cho san xuat VI v|y, chpn tgo gidng lily cd mau sic dep, chdng chiu tdt vdi siu b^nh hgi vd phii hgp vdi khi h|u Vift Nam la mgt yeu cau cap thiet hi^n nay cua cac nha chgn, tgo giong. Hi|n nay, cdng tac chgn tgo giong cay trdng nhd chi thj phan tu ngay cang dugc ap dung rdng rSi tren thg gidi vi Vift Nam Tuy nhien, Udn th6 gidi vi Vi?l Nam chua co nhiiu cac cdng trinh sii dung chi thi phan ta ISSR de chgn, tgo giong lily. Bao cao nay trinh bay vifc iing dyng chi thj ISSR de danh gia mdt sd td hgp lai lily. Nghien cihi d3 sii d\ing thdnh cong 14 chi thi ISSR de ddnh gid cdc ddng con lai cua cac Id hgp lai xa, trong dd, cac con lai 134 (dugc dgt ten mdi la L5) vi SLll(dugc d|t ten mdi li LLKll) duac xac dinh l i con lai ciia cic to hgp tuong Cmg Bel (Lilium Oriental Trumpet hybrid lily 'Belladonna") x Sor (Lilium Oriental hybrid lily 'Sorbonne') vd Sor (Lilium Oriental hybrid lily 'Sorbonne') x L (Lilium longiflorum). Vdi td hgp lai F {Lilium formolongd) vd L (Lilium longiflorum), xic djnh dugc ddng 15 (dupc dit ten mdi li LK5) li con lai thirc su cua td hgp, trong khi do ddng 14 cd kieu gen giong kieu gen ciia ddng F ngn c6 thS ket luan rang 14 la dong do phdi vd tinh ciia ddng F phit tnen thinh
Tirkhoa: chiihiphdn tu. con lai, ISSR. lalxa, lily
MO DAU
Ngdy nay, chi thi DNA da chiing minh dugc nhiing diem uu viet trong cac chucmg trinh chpn, tgo gidng cdy trdng ndng nghiep nen ngay cdng dupc dp dyng rpng rai tren the gidi va Viet Nam.
Cac chi thi DNA thudng dua vao phuong phap PCR nhu cdc chi thi RAPE), AFLP, ISSR, SSR (Staub etal, 1996; Gupta, Varshney, 2000). ISSR Id mpt chi thi dupc ap dung kha phd bien vd ky thudt sii dpng nd cd the vupt qua cdc gidi hgn md cdc ky thudt RAPD vd AFLP gdp phai (Gupta et a/..1994; Wu et a;.,l994; Meyer el ai.l993). Ky thugt ISSR dua tren phuong phap PCR nhdm nhdn cdc dogn trinh tu khdng Idp d gi&a hai trinh t\r Idp lgi xdc djnh gidng nhau nhtmg ngupc hudng, vdi moi don dupc thiet ke khoang 16-25 bp, de nhdn cdc dogn Locus khac nhau.
Hien nay, ky thugt phan tu da va dang Id rapt cdng eg huu bieu trong cdng tdc phan logi rapt cdch chinh xac hon cho cac ddng/gidng lily (Persson el ai, 1998; Wen el ai. 2001; Masumi., 2002). Ben cgnh do, mpt sd nha khoa hpc da sii dung chi thi phan tii de phan tich d giai doan sdm trong qud trinh sinh trudng va phat trien ciia thuc vat va lam ngan lai qua trinh chon gidng mdi (Karp, 2000; Orlikowska et al, 2001). Tuy nhien, cd mdt sd it cdc cdng trinh sir dyng chi thi phan tii ISSR de nghien ciiu tren lily (Guo el ai. 2011; Mengli et al.,20l2;). Hon nua, tren th6 gidi ciing it cd cong bd sii dung chi thi phan tii ISSR trong cdng tdc ddnh gia, xdc dinh con lai trgn lily. Theo nghien ciiu ciia Orlikowska et al (2001) da chi ra rang, cd the xdy ra su tiep hpp vo tinh trong qua trinh sinh san cua lily. Cac ky thuat phan tii khde nhau (Yaraagishi, 1995; Wiejacha et ai, 2001) dupc sii dung de danh gia tinh trgng ciia con lai lily thu dupc
Bui Thi Thu Huong et^i nhu la kdt qua cua lai xa. Cdng trinh cdng bd ciia Beta
el al (2007) da sir dung 11 chi thi RAPD dk danh gid 6 con lai vdi dang bd me dupc kiem tra la Lilium longiflorum x ConneetieulLas.
6 Viet Nam, cdng tac chpn tao gidng hoa lily mdi chi dupc quan tdm trong vai nam gdn day. Cac thdnh tuu ma chiing ta dat dupc trong lmh vuc nay Cling chua nhieu, chii yeu tap tnmg d lmh vuc tuy6n chpn gidng, nhdn gidng in vitro va in vivo. Trong khi do, nude ta dang sd hiiu ngudn gidng lily, loa ken dia phuang cd dgc diem tdt nhu: thich nghi vdi dieu kien khi hau trong nude, chdng chiu tdt vdi sau benh hgi. Do dd, mdt sd nhd khoa hpc da budc dau tien hdnh lai tgo nham tao ra cac gidng hoa lily, loa ken tgi Viet Nara cd radu sdc dep, huong thcrra quyen ru vdi nhirng dgc diem uu vifit khde cua hoa nhgp npi. Tuy nhien, nhiing ket qua chua cdng bd thu dupc dang dimg lgi a cho tgo ra hat lai, cii lai in vitro rad chua ddnh gid dupc cac dgc diera ndng sinh hpc, dac bipt dac diem chdt lupng hoa giai doan trudng thanh. Viec ddnh gid, kiem tra va xdc dinh
ddng con Iai gap nhidu khd khan nhu su tieu ton nhiSu thdi gian, c o i ^ siic trong trdng trot va khao nghidm. Chinh vi vay, n h i m nit ngdn cdng tac trai, chung ta cdn budc dau xac dinh sdm the h | con JR vitro vdi cap bd me bang chi thi phan tii. Vi v|y, bai bao ndy trinh bdy ket qua xdc dinh con lai ciia td hpp lai lily b i n g chi thi phdn tii ISSR,
V ^ T LIEU VA PHUONG PHAP Vdt liSu
Con lai nghi ngd ciia 3 td hpp lai xa chi Lilhm dupc cung cap bdi Vien Nghien cihi Rau qud. Trong dd, 100134 (viat tit la 134) Id con lai giffa Bel (Lilium Oriental hybrid lily 'Belladonnd) vaSor (Lilium Oriental hybrid lily 'Sorbonne'); SLll la con lai giita Sor (Lilium Onental hybrid lify 'Sorbonne'} vd L (Lilium Oriental hybrid lily Lilium longiflorum): 14, 15 la con Iai giira F (Liliim formolongo ) va L (Lilium longiflorum).Csy bo me
cd dac diem hinh thai vd phdn logi theo Bang 1.
BSngl. Phan logi va dgc diem hinh thai cay i^m bd, me trong nghien c u u .
STT Ten ggi Ky hi^u Dgc diem d i e t n m g hoa Ngudn goc
Belladonna Bel Lilium . longiHorum Lilium p iormolongo
- Trang hoa mau hong, cd c h i m fid, vi4n trang.
- Hoa h u d n g len tren, cd mui rattho'm.
- Trang hoa mau vang ti/ai - Hoa quay ngang, cd mui r i t thorn.
- Trang hoa mau IrSng.
- Hoa quay ngang, cd mui thom nhe.
- Trang hoa mau trSng.
- Hoa hirdng len tren, c» mui thom nhg
Uly Phmmg dong (Oriental liiy) Lily lai loa ken (Oriental Trumpet lily) Loa ken (Trumpet lily) Loa ken (Trumpet lily)
Phuorng p h a p
Phuang phdp tdeh chiit DNA tdng so
DNA lily dupc tdeh chiet va tinh sgch theo phucmg phap CTAB cua Doyle vd cpng su cd cdi tien (Doyle et a/.,1987). Sau do DNA dupc do ndng dp bdng mdy NanoDrop Lite (Thermo Scientific) vd pha loang ra ndng dp sir dung.
Phifongphap PCR- ISSR
Phdn ling PCR - ISSR dupc thuc hien tren may Veriti™ 96 well thermal Cycler (Applied Biosystems, USA), vdi tdng the tich la 15 [iV mau
gdm nhung thdnh phin la DNA (100 ng/ul) - 1,8 |il;
mdi RAPD (10 pmol) - 1,2 ^il. Master Mix 2X - 7^S pl; ddHiO - 4,5 pl; theo chucmg trinh da cai dat san nhu sau:
Bucfcl: 94''C trong 4 phiit; budc 2: 92''C trong I phut; budc 3: Ta''C (nhiet dp bat cap tirng mdi) troi^
I phut; budc 4: 72''C trong 1 phut; budc 5: lap lgi 35 chu ky tli budc 2 dgn budc 4; budc 6: 72''C trong Id phut; budc 7: giQ nhiet dp 10''C.
Trong nghien ciiu ndy cac i trinh bay d Bdng 2.
i sii dung duct
516
Tgp chi Cdng nghe Sinh hpc 12(3): 515-522,2014
Bdng 2. Tdn vd trinh t ^ cac mdi ISSR s i i dyng trong n g h i ^ c
Trinh tif m&i T r i n h t v m 6 i
ISSR 4 ISSR 5 ISSR 6 ISSR 7 ISSR 8 ISSR 15 ISSR 51
(CCG)6 (CTC)fl (GGC)e (GAA)fl (TG)8GA (CT)aT (GA)aA
ISSR 52 ISSR 53 ISSR 55 ISSR 56 ISSR 59 ISSR 62 ISSR 69
(CT),G (CA)sA (AC)flT (AC)flG (GA)aCT CTC(AG)7 (GGGTG)3
Phuang phdp phdn tich so li?u ISSR
Sau khi cd ket qua dien di tren gel agarose 0,8%, chiing tdi tien hanh so sanh cac bdng vgch DNA cua con lai so vdi bo va me c6 mat hay vdng mat d ban di^n di. Cy thi Id, nlu cd bang DNA cd kich thudc nhat dinh xudt hi^n bdt ki ddng d con lai hay ddng lam bd (ho§c m^) thi ky hieu "+"; cdc gieng cdn lgi khong xuat hipn thi ky hieu la "-". D^a vdo miic do xuat hi^n ciia cac bang nay chiing ta co the danh gid dupc con lai dupc xem xet cd phdi Id con lai thuc su khdng vd con lai dd cd quan hp vdi bd ho|ic me chiing. N I U con lai nghi ngd nao cd bdng vgch cua ca b6 lEn m^ thi chde chdn dd Id con lai cita td hpp lai dd. N^u con lai ndo hodn todn gi6ng bo hodc me d tat cd cac chi thi nghien ciiu thi con lai do la tu thu ciia ddng bd hodc rap tuong img.
Tien hdnh xii ly sd Iifu vd Idp bieu do phan logi:
Cdc sd li§u cdc vgch bang xudt hifn thu dupc tir bang difn di dupc dua vao xir ly theo chuong trinh NTYSYS 2.2 (Rohlf, 2000) d£ Igp bifu dd phan
nhdm 2 chieu dua tren phdn tich PCA (Principal Coordinate Analysis) qua do se lap bieu dd phan nhdm dua tren khoang each di truyen 2 chieu giira con lai vd ddng bd me.
K £ T QUA VA THAO LUAN Ket qud tdeh chiet DNA
DNA dupc taeh chilt vd tinh sgch s5 dupc do ndng dp vd df sgch (OD2eanso) bing mdy do NanoDrop. Ket qua dupc dil hifn trong bdng sau:
Tren Bang 3 cd the thay rang DNA lily dupc taeh chiet vdi dp sgch cao vdi ty If OD26(i/280 nam trong khoang 1,84 den 1,93. Ndng dp ciia cdc raau DNA cung rdt cao nam trong khoang 426,0 den 797,2 ng/ul. DNA dupc tdeh chilt dii chat lugng de tiln hanh phdn iing PCR-ISSR sau khi da pha loang DNA den ndng dd la 100 ng/ul.
Bdng 3. N6ng dd vA dd sgch cda 8 m i u DNA lily thugc 3 to hgp lai xa
OD260;280 1,87 1,90 1,89 1,90 1.93 1,93 1,84 1.86
Ham iirgng (ng/ul) 621,6 487.6 797.2 426.0 668.3 490,1 596,4 439,7
Biii Thi Thu Hirong tf rf Ket qua xsc d|iih con lai bdng chi tlii ISSR
De nghien ciiu quan he di truyen giira cdc gidng lily, 14 radi ISSR cd ten va trinh tu tai Bdng 2 da dupc sii dung. Sau khi hoan thdnh tiing phan ung PCR, san phdm dupc dien di tren gel agarose 1,2 % de phdn tich da binh DNA ciia cac mau nghien ciiu, vi du nhu Hmh I, Hinh 3.
Kit qua xac dSiiA con lai 134 cua id hgp Bel va Sor Hinh l a the hifn k i t qud difn di san phdm PCR cua td hop lai Bel x Sor vd con lai 134 vdi mdi ISSR 51. Ta cd thi thdy, con lai 134 cd 3 bang vach DNA dupc nhan len vdi kich thudc khoang 500, 700 vd 1000 bp. Trong do, bang d vi tri 500bp teiing vdi bang
SOObp cua ddng Bel, bang 700bp trimg voi bi«|
700bp cua ca Bel va Sor nfn khdng cd y nghla d U gia su da hinh cdc bang DNA, bdng lOOObptritaj^
bang cua Sor. Tuong tu. Hinh lb la anh difn d i £ i phdm PCR-ISSR ciia td hop Bel x Sor va con b i ^ vdi mdi ISSR 55. Con iai 134 cd 4 bdng v^ch DNA dupc nhdn len vdi kich thudc khodng 600, 700,800ri 900 bp; trong dd, bang d vi tri 600bp vd bdng 700^
triii^ vdi bang 600bp vd bang 7(X)bp ciia ddng Bd, bang SOObp trimg vdi bang 800 bp cua Sor, bdi^ 901 bp Id bang chung cua cd td hpp.
Phan tich tdng hpp su sai khac cac bang DNA sdn phdm PCR-ISSR cua to hpp nay vdi 14 mfli ISSR dugc the hifn trong Bang 4.
134^ B e l
Hinh 1 . Anh €t^ di san phdm PCR4SSR to hpp Sor x Bel va con lai 134 v d i md* ISSR 51 (Hinh a) va ISSR 55 (HllA b).
134: con lai cua td hgp Bel va S o r M: marker I M J
Bang 4 . Phan tich da htnh DNA cua con lai so vdi ddng bit, me cua td hop Sor va Bel.
c c a . t c i r . c c v c ' B a -
in ta o) CO W ( o < o ( 0
C O C 0 C 0 ( 0 U > ( ( } V ) ( 0
Bang DNA (bp)
Ghi chu: (+): cac l>ang DNA xuat hien; (-): cac bang Dh4A ktidng xuat hien, (*) khong cho ket q j a da hin
Tgp chi Cong nghi Sinh hpc 12(3): 515-522,2014 Tren bang 4, ta cd the thdy rang trong 14 mdi da sir dung df ddnh gid thi cd 2 mdi khdng cho ket qud da hinh la radi ISSR8 va ISSR56. Tdng sd bang d con lai thu dugc Id 23 bdng DNA, trong do c6 14 bang gidng cua ddng Bel va 9 bang gidng ddng Sor.
Nhu vdy, 134 mang mpt sd bang gidng ca hai ddng Bel va Sor, cd bang gidng vdi ddng ldm me (Bel) raa khdng xuat hien d ddng lam bo (Sor); ngupc lgi cd bang gi^ng vdi giong lam bd ma khdng xuit hifn a giong lara me. Qua ddy, budc ddu cd the ket luan dupc rMig ddng 134 chinh la con lai ciia Bel va Sor.
Tien hanh phan tich sd lieu san pham PCR - ISSR cua td hpp trfn ciiug con lai bang phdn mem NTYSYS 2.2 df lap ban dd 2 chilu da so sanh dugc quan hf di truyen cua con lai dd vdi bd, me n h u d Hinh 2.
O Hinh 2 ta CO thf thdy con lai 134 nam gan ddng Bel hon so vdi gidng Sor, chimg td ddng 134 (ten goi mdi la L5) cd quan he di truyen gan ddng Bel hon so vdi gidng Sor khi phdn tich bang chi thi tinh trpi ISSR.
-0.34
-0 48 -0.1 Hinh 2. Bieu dd tga dg 2 chieu cua to hpp lai
0.10 Dim-1 X Sor va con lai 134.
Xdc dinh con lai SLII cua to hgp Sor va L vd con laiI4,15 cua td hgp FvdL
Hinh 3 la anh dien di sdn phdm PCR-ISSR ciia con lai S L l l cua td hgp lai Sor va L; con lai 14 , 15 ciia td hpp lai L vd F,
d hinh 3a ta thdy con lai S L U cd cac bang vgch hoan toan trung vdi cdc bang vgch gidng vdi gidng Sor, Tuy nhifn, d Hinh 3b ta thdy, con lai SLII cd 2 bang khoang 600 va 900 bp, trong do bdng 600 bp giong vdi bang ddng L va bang 900 bp gidng vdi bdng ciia gidng Sor. N h u vdy, S L I I (ten gpi radi la LLKl I) co the budc dau xac djnh Id con lai ciia to hgrp nay.
Trong td hpp lai F va L, d Hinh 3a, con lai 15 cd 3 bang la 600, 700 va 900 bp trong do co bang 600 l ^ gidng vdi bang ciia ddng L, bang 700 bp la bang xudt hifn d cd td hgp nfin khdng co ^ nghia danh gia sir da dgng DNA, bang khodng 900 bp gidng vdi bang d dong F. Mac dii, khi kiem tra td hpp lai F x L vdi mdi ISSR55 (Hinh 3b) ta cd the thay rang, cac bang ciia con lai 15 gidng vdi cac bang cua ddng L, tuy nhien van cd the budc ddu xac dinh 15 la con lai. Dieu nay dupc chung minh rd hon khi phdn tich vdi tdt ca cdc chi thi ISSR da sii diu^ (Bang 5). Con lai 14 cd lat ca cac bang DNA gidng vdi bang cua ddng F (Hmh 3a, b). Nhu vay, ta cd flie budc dau cho rang, ddng 14 cd ftif la ddng do phdi vd tinh ciia F tgo thanh.
519
Hinh 3. Anh dien di san phSm PCR-ISSR td hgp Sor vd L. a: L vg F va cac con lai vdi mdi ISSR 7 va ISSR 55, b. SL a lai cua td hgp Sor va L. 14.15 con lai cua td hgp L vd F; M: marker Ikb
Khi kiem tra 14 chi thi ISSR vdi hai td hpp thi cd 14 chi thi dfu cho cdc bdng vgch giiip xac dinh con lai.
Trong dd, vdi td hpp lai xa Sor va L thi co tdt ca 33 bang thu dupc d con lai SLl I thi cd 22 bang gidng vdi gidng Sor va 11 bang gidng vdi ddng L. Vdi td hgp lai xa F va L, con lai 14 cd tat ca cac bang gidng vdi bdng cua ddng F (ddng ldm me) nen cd the ket luan rang 14 chinh Id do phdi vd tinh phat trien thanh. Vdi td hpp lai F vd L thi ddng 15 (vdi ten mdi la LK5) Id dong lai thuc su giiia F vd L, trong do, thu dupc tdng s6 29 bang
nhgn bilt con lai vdi 22 bdng gidng vdi ddng L v4 7 bang gidng ddng F (Bdng 5).
Tii kft qua phdn tich ta cd the lap bteu dS 2 chifu the hifn mdi lien hf cua con lai vdi cdc d p bi, rap ciia chung:
Tren hinh 4 ta cd the thay, khi danh gid con lii vdi cbi thi tfnh trpi ISSR d td hgp lai Sor va L thi con lai S L I I ndm gdn ddng Sor hon vdi L. Tuong tu, con lai 15 nam gan ddng L b a n so vdi ddng F.
Bang 5. Phan tich da hinh DNA cua con lat so vdi ddng bd, m^ c 6 hgp Sor v^ L; F vd L.
Tgp chi Cong nghe Sinh hge 12(3): 515-522,2014 0.58
-0.34
-0.48 . -0.19 0.10 Dim-I
Hinh 4. Bi^u dd tga dd 2 chi^u cua to hgp lai Sor vd L; td hgp F va L
KET L U A N
Da danh gia thanh cdng con Iai ciia 3 td hgp lily lai xa bdng 14 chi thi tinh trdi ISSR. Trong dd, con lai 134 ( L 5 ) dugc xac dinh Id con Iai cita Belladonna;
S L l l ( L L K l I ) Id con lai ciia td hpp lai gi&a Hly Sor vd loa kfn L; vdi td hgp lai loa kfn F vd L vdi nhau, xdc dinh dugc ddng 15 (LK5) la con lai th\rc su cda td hpp, trong khi do ddng 14 la ddng do phdi vd tinh ciia ddng F (ddng me) phat triln thanh.
LM cam on: Nghien cicu ndy dirge ho Ira mgl phdn kinh phi cita de tdi cdp bg "Nghiin edu ung dung cong ngh? sinh hoc tien tien aia Hd Lan trong chpn.
tgo. nhdn giong vd diiu khien ra hoa chi Lilium (Lily, Loa ken) & Viit Nam"do Vien Nghien ciru Rau qud lam chil tri. Chung toi cSng xin cdm an cdc cdn bg Phong Cong ngh? ADN dng dung vd Phong Cong ngh? le bdo Thgc vdt - Vi?n Cong nghi smh hpc.
Vi?n Hdn ldm Khoa hpc vd Cong nghi Viet Nam dd giiip dd ve mdy moc. thiet bi vd chuyen mon de thgc hi?n nghien cmi ndy.
TAI LIEU THAM KHAO
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I N I T I A L S T U D Y A N D A P P L I C A T I O N O F I S S R M A R K E R S T O D E T E C T I N G HYBRIDS O F LILIUM G E N U S I N V I E T N A M
Bui Thi Thu Huong^'^^ Ho Manh Tuong', Le Van Son', Le Tran Binh', Dang Van Dong', Trinh Khac Quang^
'institue of Biotechnology, Vietnam Academy of Science and Technology
^ Vietnam National University ofAgricuilture, Ministry of Agriculture and Rural Development
^Fruits and Vegetables Research Institute, Vietnam Academy o Agricultural Sciences
SUMMARY
Lily IS one of the most favourite flowers which have high value producing, nice color, fragance and prefrered by consumers In Vietnam, lilies have been planted since many years ago in Dalat, Lam Dong province and get increased m quantity and scale, recently. Lilies have been also grown in the North of Vietnam since 2001 with small scale . Viemam and many other countries have not produced tily bulbs, so they have to import from Netherland at quanity of about 60 million lily bulbs for production. Breeding lily varieties wilh beautiful color, fragrance and suitable for the climate conditions in Vietnam is nessesary trend for the breeders.
Nowaday, plants breedmg by using molecular markers is getting more and more applicable in the worid and in Vietnam. However, there are few sUidies using ISSR markers to breed lily varieties. This smdy presents the evaluation some lily hybrids combinations by using 14 ISSR markers. The results showed that the lines 134 (L5) and SLll(LLKll) have been identified as the hybrids of the respective combination of Bel (Lilium Oriental Trumpet hybrid lily 'Belladonna') x Sor (Lilium Onental hybrid lily 'Sorbonne') and Sor (Lilium Oriental hybrid lily 'Sorbonne') x L {Lilium longiflorum). Ofthe combinations of F (Lilium formolongo) X L(Lilium longiflorum), I5(LK5) line is actually identified as a hybrid of the combination , while 14 line is evaluated to have the same genotype as F line. Therefore, it is concluded that 14 developed from an asexual embryo in the F line.
Keywords . breeding, ISSRs. lilium. molecular markers
• Author for correspondence: E-mail: btlhuonghp(fl>,gmail com 522
