Nghiên cứu tính đa dạng di truyền của một số giống ngô (ZEA MAYS L)

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I HC THI NGUYN TRNG I HC S PHM --------------------------------
TRN TH NGC DIP
NGHIN CU TNH A DNG DI TRUYN CA MT S GING NG (ZEA MAYS L.)
lun vn thc s sinh hc
Thi Nguyn: 2009
S ha bi Trung tm Hc liu i hc Thi Nguyn
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I HC THI NGUYN TRNG I HC S PHM ------------------------------
TRN TH NGC DIP
NGHIN CU TNH A DNG DI TRUYN CA MT S GING NG (ZEA MAYS L.)
Chuyn ngnh: Di truyn hc M s: 60.42.70
LUN VN THC S SINH HC
Ng-i h-ng dn khoa hc: PGS.TS CHU HONG MU
Thi Nguyn: 2009

LI CAM OAN Ti xin cam oan y l cng trnh nghin cu ca ring ti. Cc s liu, kt qu nghin cu trong lun vn l trung thc, cha c ai cng b.
Tc gi
Trn Th Ngc Dip

LI CM N Ti xin by t lng bit n ti PGS. TS Chu Hong Mu tn tnh hng dn, ch bo v to mi iu kin gip ti hon thnh lun vn ny. Ti xin cm n TS. Nguyn V Thanh Thanh - B mn Khoa hc s sng - Trng i hc khoa hc i hc Thi nguyn tn tnh gip ti trong qu trnh thc hin cc th nghim nghin cu ca ti. Ti xin chn thnh cm n Lnh o Trng i hc S phm - i hc Thi nguyn, Ban ch nhim Khoa Sinh KTNN v cc thy c gio, cn b trong khoa to mi iu kin gip ti trong qu trnh hc tp v hon thnh lun vn. Ti xin chn thnh cm n s ng vin, khch l v to mi iu kin ca Ban gim hiu Trng i hc Hng Vng, Ban ch nhim khoa Nng Lm Ng, cng cc ng nghip, gia nh v bn b to mi iu kin v gip ti trong sut qu trnh hc tp v lm lun vn. Tc gi
Trn Th Ngc Dip

MC LC Trang Li cam oan......... Li cm n Mc lc..... Nhng ch vit tt. Danh mc cc bng........... Danh mc cc hnh........... M U.......... Chng 1. TNG QUAN TI LIU.... 1.1.CY NG.............. 1.1.1.Ngun gc v phn loi cy ng.. 1.1.2.c im nng sinh hc ca cy ng............... 1.1.3.Vai tr cy ng trong nn kinh t. 1.1.4.c im ha sinh ht ng............... 1.1.5.Tnh hnh sn xut ng trn th gii v Vit Nam............ 1.1.5.1.Tnh hnh sn xut ng trn th gii......... 1.1.5.2.Tnh hnh sn xut ng Vit Nam...... 1.2. TNH HNH NGHIN CU TNH A DNG DI TRUYN THC VT......................................... 1.2.1.Mt s phng php sinh hc phn t s dng trong nghin cu quan h di truyn thc vt......................... 1.2.1.1.K thut RFLP (Restriction Fragment Length Polymorphisms a hnh di cc on ct gii hn)
1.2.1.2. K thut AFLP (Amplified Fragment Length Polymorphism a
i ii iii vi vii viii 1 3 3 3 3 5 7 8 8 12
13
13
14

hnh di cc on c nhn bn chn lc).
14
1.2.1.3. K thut SSR (Simple Sequence Repeat trnh t lp li n gin).. 1.2.1.4.Bn QTL (Quantiative Trait loci) 1.2.1.5. K thut RAPD (Random Amplified Polymorphic DNA)... 1.2.2. Nghin cu s a dng di truyn thc vt bng k thut RAPD............... 1.2.3.Tnh hnh nghin cu s a dng di truyn ca ng bng k thut RAPD....... 1.3. NHN XT CHUNG............... Chng 2 . VT LIU V PHNG PHP...... 2.1.VT LIU NGHIN CU.. 2.1.1.Vt liu thc vt... 2.1.2.Ho cht............... 2.1.3.Thit b......... 2.2. PHNG PHP NGHIN CU... 2.2.1.Phng php ha sinh.. 2.2.1.1.Xc nh hm lng lipid.. 2.2.1.2.Xc nh hm lng protein............. 2.2.1.3.Xc nh hm lng ng tan..................................................... 2.2.2.Phng php sinh hc phn t......................................................... 2.2.2.1.Phng php tch DNA t l non ca ng................................... 2.2.2.2.Phng php xc nh hm lng v tinh sch DNA tng s............. 2.2.2.3.Phn ng RAPD............................................................................ 2.2.2.4.Phng php x l kt qu v tnh ton s liu............................ Chng 3. KT QU V THO LUN.................................................. 3.1. C IM HNH THI, HA SINH HT CA CC GING NG NGHIN CU.................................................................................... 31 15 17 17 20 22 24 25 25 25 25 26 26 26 26 26 27 27 27 28 29 30 31

3.1.1.c im hnh thi ca 14 ging ng nghin cu................................ 3.1.2.Hm lng protein, lipid, ng ca 14 ging ng nghin cu.......... 3. 2. PHN TCH TNH A HNH DNA BNG K THUT RAPD... 3.2.1.Kt qu tch chit DNA tng s t l ng....................................... 3.2.2.Kt qu nghin cu a hnh DNA bng k thut RAPD................. KT LUN V NGH.......................................................................... CNG TRNH CNG B LIN QUAN N LUN VN.............. TI LIU THAM KHO...................................................................... 31 32 35 35 37 50 51 52

NHNG CH VIT TT Amplified Fragment Length Polymorphism (Tnh a hnh chiu di cc phn on c nhn bn) ASTT CS DNA dNTP EDTA ISSR Kb LEA p sut thm thu Cng s Deoxyribonucleic acid Deoxyribonucleotit triphotphat Ethylene Diamin Tetraaxetic Acid Inter Simple Sequence Repeats Kilobase Late Embryogeneis Abundant protein (Protein tng hp vi lng ln giai on cui ca qu trnh pht trin phi) PCR RAPD Polymerase Chain Reaction (Phn ng chui polymerase) Random Amplified Polymorphism DNA (Phn tch ADN a hnh c nhn bn ngu nhin) RFLP Restriction Fragment Length Polymorphism (Phn tch chiu di cc phn on ADN ct hn ch) SDS Sodium Dodecyl Sulphat SDS-PAGE Phng php in di trn gel polyacrylamid c cha SDS SSR STS TBE TAE TE Tris Simple Sequence Repeats Sequense Tagged Site Tris - Boric acid - EDTA Tris - Acetate - EDTA Tris - EDTA Trioxymetylaminometan
AFLP

DANH MC CC BNG
Bng 1.1 1.2 1.3
Tn bng Thnh phn ho hc ca ht ng v go (Phn tch trn 100) D bo nhu cu ng th gii n nm 2020.. Tnh hnh sn xut ng ca mt s khu vc trn th gii giai on 2005 2007...
Trang 8 9
10
1.4
Tnh hnh sn xut ng ca mt s quc gia trn th gii nm 2007 11 13 25
1.5 2.1 2.2
Tnh hnh sn xut ng Vit Nam t nm 2004 n nm 2006 c im 14 ging ng nghin cu... Trnh t cc nucleotide ca 10 mi RAPD s dng trong nghin cu.
29 30 31 33 36 38 47 48
2.3 3.1 3.2 3.3 3.4 3.5 3.6
Thnh phn phn ng RAPD. c im ca 14 ging ng np a phng. Hm lng protein, lipid, ng trong ht ca 14 ging ng... Ph hp thu DNA bc sng 260nm v 280nm. S phn on DNA xut hin tng ging ng nghin cu.. T l phn on a hnh khi s dng 10 mi RAPD. H s tng ng di truyn ca 14 ging ng np

DANH MC CC HNH Hnh 3.1 3.2 3.3 3.4 Tn hnh Hnh dng ht ca 14 ging ng..
Hnh nh in di DNA tng s ca 14 ging ng
Trang 32 35 37
Ph hp th DNA ca ging SLV o bc sng 260 nm. Hnh nh in di sn phm RAPD vi mi M1 ca 14 ging ng.
39
3.5
Hnh nh in di sn phm RAPD vi mi M2 ca 14 ging ng. 40
3.6
3.7
3.8
3.9
3.10
Hnh nh in di sn phm RAPD vi mi RA159 ca 14 ging ng. 44
3.11
Hnh nh in di sn phm RAPD vi mi UBC23 ca 14 ging ng. 46 49
3.12
Biu m t quan h di truyn ca 14 ging ng

M U
1. L do chn ti Cy ng c tn khoa hc l Zea mays L. v c ngun gc t Trung M. Ng l cy lng thc quan trng trong nn kinh t ton cu. cc nc thuc Trung M, Nam v Chu Phi, ngi ta s dng ng lm lng thc chnh. Khng nhng th, ng cn l cy cung cp thc n chn nui quan trng nht hin nay: 70% cht tinh trong thc n tng hp ca gia sc l t ng (Ng Hu Tnh, 2003) [18]. Ng khng ch cung cp lng thc cho con ngi, pht trin chn nui, ng cn l nguyn liu cho ngnh cng nghip ch bin trn ton th gii. Hin nay, din tch ng trn th gii vo khong 135 - 140 triu ha, vi sn lng trung bnh l 600 - 700 triu tn. Vit Nam, ng l cy lng thc quan trng th hai sau la ca nng dn vng trung du v min ni pha Bc ni chung v cy lng thc chnh ca ng bo dn tc thiu s vng cao ni ring [1]. Trong nhng nm gn y sn xut ng Vit Nam tng ln nhanh nh s thc y ca ngnh chn nui v cng nghip ch bin. c bit t nhng nm 1990 tr li y, din tch, nng sut v sn lng ng tng lin tc l nh ng dng nhng tin b khoa hc k thut mi vo sn xut m tiu biu l a ng lai vo trng trn din tch rng. Cc ging ng nc ta hin nay rt phong ph gm cc ging ng nhp ni, ging lai to, ging tng hp, ging t bin v cc ging ng a phng [20]. Bn cnh cc ging ng lai c nng sut cao ang c trng ph bin nhiu vng trong c nc, th cc ging ng a phng tuy c nng sut thp nhng cht lng ht cao, cht lng ng do, thm, ngon v chng chu su bnh tt. Hin nay, c rt nhiu phng php nghin cu s a dng di truyn ca cc ging cy trng ni chung v cy ng ni ring nh RFLP, AFLP, 1
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SSR, STS, RAPD,... Cc phng php ny khc phc c nhc im ca cc phng php chn ging truyn thng bi nh gi c h gen ca cy trng. Nhng nm gn y, din tch trng cc ging ng a phng ngy cng c xu hng gim, nhiu ging ng np qu him s b mt dn. Nh vy, vic su tp v nghin cu cc ging ng np a phng gp phn bo tn ngun gen cy ng l rt cn thit. Nghin cu s a dng di truyn mc DNA v c im ha sinh giai on ht l c s khoa hc xut vic chn nhng ging ng c nng sut cao v cht lng tt gp phn bo tn, pht trin ngun gen cy ng. T tuyn chn ging ng thch hp lm vt liu chn ging l nhng vn rt c quan tm nghin cu. Xut pht t l do trn, chng ti tin hnh thc hin ti: Nghin cu tnh a dng di truyn ca mt s ging ng (Zea mays L.) 2. Mc tiu nghin cu - nh gi cht lng ht ca mt s ging ng np a phng (Zea mays L.) - Kho st s a dng v mi quan h di truyn ca 14 ging ng bng k thut RAPD. 3. Ni dung nghin cu - Phn tch c im hnh thi, khi lng v kch thc ht ca mt s ging ng np a phng. - Xc nh hm lng lipid, protein, ng trong ht ca cc ging ng nghin cu. - Phn tch s a hnh DNA c nhn bn ngu nhin, xc nh mc sai khc trong cu trc DNA h gen ca cc ging ng nghin cu. - Thit lp mi quan h di truyn ca 14 ging ng.
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Chng 1 TNG QUAN TI LIU
1.1. CY NG 1.1.1. Ngun gc v phn loi cy ng Cy ng (Zea mays L.) thuc chi Maydeae, h ha tho Gramineae, c ngun gc t Trung M. Ng c b nhim sc th (2n=20). C nhiu cch ngi ta phn loi ng, mt trong cc cch l da vo cu trc ni nh ca ht v hnh thi bn ngoi ca ht. Ng c phn thnh cc loi ph: ng rn, ng rng nga, ng np, ng ng, ng n, ng bt, ng na rng nga. T cc loi ph da vo mu ht v mu li ng c phn chia thnh cc th. Ngoi ra ng cn c phn loi theo sinh thi hc, nng hc, thi gian sinh trng v thng phm [12]. C rt nhiu gi thuyt v ngun gc ca ng ti chu M nh ng l sn phm thun dng trc tip t c ng (Zea mays ssp. parviglumis) mt nm Trung M, c ngun gc t khu vc thung lng sng Balsas min nam Mexico. Cng c gi thuyt khc cho rng ng sinh ra t qu trnh lai ghp gia ng thun ha nh (dng thay i khng ng k ca ng di) vi c ng thuc on Luxuriantes. Song iu quan trng nht n hnh thnh v s loi ph, cc th v ngun d hp th ca cy ng, cc dng cy v bin dng ca chng to cho nhn loi mt loi ng cc c gi tr ng cnh la m v la nc [12]. 1.1.2. c im nng sinh hc ca cy ng C quan sinh dng ca ng gm r, thn v l lm nhim v duy tr i sng c th. Ht c coi l c quan khi u ca cy. Sau khi gieo ht, ng pht trin thnh mm. Cy mm ch yu s dng ngun dinh dng cha trong ni nh ht. B phn pha trn ht pht trin ln mt t gm c tr gia l mm. Phn nh tr l mm c mu bao 3

l mm, t pht sinh bao l mm v bn trong bao l mm l thn l mm. Trn trc ca cy mm, mt u hnh thnh r cy mm, sau pht trin thnh r chnh, t r chnh hnh thnh cc r ph. Ng l cy c r chm tiu biu cho b r cy ha tho. H r c ba loi: r mm, r t v r chn king. R t gip cho cy ht nc v cc cht dinh dng. R chn king mc xung quanh cc t phn thn st gc trn mt t, r ny gip cy chng , ng thi cng tham gia vo ht nc v thc n cho cy. S lng r, s lng r v chiu di r khc nhau mi ging. Thn ng thng pht trin mnh, thng cng dng bn chc. Thn chia lm nhiu ging, cc ging nm gia cc t, cc ging di v to dn t di ln. L ng mc t mt trn t v mc i xng xen k nhau. ln v s l ng dao ng t 6 n 22 l ty thuc vo ging v iu kin t nhin. L ng trng thnh bao gm cc b phn: b l, phin l v tha l. Bp ng pht sinh t mm nch l trn thn, s mm nch l trn cy ng nhiu, nhng ch 1-3 mm nch trn cng pht trin thnh bp. Tu thuc vo ging, iu kin sinh thi, chm bn, mt , ma v m t l cy 2-3 bp, s ht trn bp, v tr ng bp, thi gian phun ru, tr cc khc nhau. Ht ng thuc loi qu dnh gm 4 b phn chnh: v ht, lp alron, phi v ni nh. Pha di ht c gc ht gn lin vi li ng. V ht bao bc xung quanh, mu sc v ht ty thuc vo tng ging, nm sau lp v ht l lp alron bao bc ly ni nh v phi. Ni nh l thnh phn chnh 70-78% trng lng ht, thnh phn ch yu l tinh bt, ngoi ra cn c protein, lipid, vitamin, khong v enzyme nui phi pht trin. Phi ng ln (chim 8-15%) nn cn ch trng bo qun [12]
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Mi mt giai on sinh trng, cy ng yu cu v iu kin sinh thi khc nhau. Trong iu kin m bo v m , xy v nhit thch hp cho ng ny mm nhanh sau khi gieo. Nhit ti thiu cho ht ny mm t 8 120C, nhit ti a cho ht ny mm t 40 450C, nhit ti thch t 25 280C. cc thi k sinh trng khc nhau th s ht cht dinh dng cng nh yu cu v dinh dng ca ng cng khc nhau: thi k u cy ng ht cht dinh dng chm, thi k t 7 - 8 l n sau tr 15 ngy ton b cc b phn trn mt t cng nh cc b phn di mt t ca cy ng tng trng nhanh, cc c quan sinh trng pht trin mnh, lng tinh bt v cht kh tng nhanh. y l giai on cy ng hp thu cht dinh dng ti a (bng 70 - 90% dinh dng c vng i cy ht). thi k ny nu cy thiu nc v cht dinh dng s lm gim nng sut t 10 - 20%. Trong cc yu t dinh dng th m l nguyn t dinh dng quan trng bc nht ca cy ng (L c Bin,1986) [2]. 1.1.3. Vai tr cy ng trong nn kinh t Ng lm lng thc cho con ngi: Ng l cy lng thc nui sng gn 1/3 dn s trn ton th gii, tt c cc nc trng ng ni chung u n ng mc khc nhau. Ton th gii s dng 21% sn lng ng lm lng thc cho ngi. Cc nc Trung M, Nam v Chu Phi s dng ng lm lng thc chnh. Cc nc ng Nam Phi s dng 85% sn lng ng lm lng thc cho ngi, Ty Trung Phi 80%, Bc Phi 42%, Ty 27%, Nam 75%, ng Nam v Thi Bnh Dng 39%, ng 30%, Trung M v cc vng Caribe 61%... Nu nh Chu u khu phn n c bn l: bnh m, khoai ty, sa; Chu : cm (go), c, rau xanh (canh) th chu M La Tinh l bnh ng, u v t. V vy, trn phm vi th gii, ng vn cn l cy lng thc rt quan trng, v ng rt phong ph cc cht dinh dng hn la m v go. 5

Ng lm thc n gia sc: Ng l thc n gia sc quan trng nht hin nay. Hu nh 70% cht tinh trong thc n tng hp l t ng, iu ph bin trn ton th gii. Ngoi vic cung cp cht tinh, cy ng cn l thc n xanh v chua l tng cho i gia sc. Nhng nm gn y cy ng cn l cy thc phm, ngi ta dng bp ng bao t lm rau cao cp. S d, ng rau c dng v n sch v c hm lng dinh dng cao. Cc th loi ng np, ng ng (ng ngt) c dng lm thc n ti (luc, nng) hoc ng hp lm thc phm xut khu. Ng l nguyn liu chnh cho cc nh my thc n gia sc tng hp, ng cn l nguyn liu cho cc nh my sn xut ru, tinh bt, bnh ko Ngi ta sn xut ra cc mt hng khc nhau cho cc ngnh cng nghip lng thc - thc phm, cng nghip dc v cng nghip nh. Ng cng l hng ho xut khu. Hng nm lng ng xut khu khong 70 triu tn. l ngun li ln ca cc nc xut khu. Cc nc xut khu chnh l M, Php, Argentina, Trung Quc, Thi Lan. Cc nc nhp chnh l Nht Bn, Hn Quc, Lin X c, Chu Phi, Mexico [13]. Ng va l cy lng thc, va l cy thc n cho gia sc. Chnh v vy din tch trng ng trn th gii tng khng ngng. Nm 1979 din tch trng ng ch t khong 127 triu ha vi tng sn lng l 475,4 triu tn, n nm 2007 din tch trng ng t 145,1 triu ha vi sn lng 705,3 triu tn (theo s liu thng k ca FAO, 2008). Vit Nam, cy ng c trng cch y khong 300 nm v c trng trn nhng iu kin sinh thi khc nhau ca c nc. Hm lng cht dinh dng ca ng ty thuc vo tng ging, c bit l cc ging ng np a phng tuy nng sut khng cao nhng cht lng ca ht ng tt ph hp vi th hiu ca ngi tiu dng. V vy, vic nghin
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cu c im hnh thi, ha sinh ca cc ging ng gip chn c cc ging ng c nng sut cao v cht lng tt p ng cho cng tc ging v nhu cu s dng ca con ngi. 1.1.4. c im ha sinh ht ng Cc cht trong ht ng d b ng ha nn c gi tr dinh dng cao. Ht ng cha tinh bt, lipid, protein, ng (chim khong 3,5%), cht khong (chim khong 1 2,4%), vitamin (gm vitamin A, B1, B2, B6, C v mt lng rt nh xenlulo (2,2%). Ht ng cha phn ln tinh bt, hm lng tinh bt trong ht thay i trong gii hn 60 - 70%. Hm lng tinh bt ng t nhiu hn ng np (68% so vi 65%). Tinh bt tp trung ch yu ni nh v c chia thnh hai dng tinh bt l tinh bt mm (tinh bt bt) v tinh bt cng (tinh bt sng hay tinh bt phal). Hm lng lipid cao th hai trong cc loi ng cc sau la mch, n chim khong (3,5 7%) v ph thuc vo tng ging, iu kin t nhin. Lipid c tp trung nhiu phi v mng alron. Du ng cha n 50% acid linoleic lin kt vi glyxerit, acid oleic, panmitic, ricinic. Hm lng lipid l mt ch tiu quan trng nh gi cht lng ht [9]. Protein ca ng c chia thnh 3 dng chnh: protein hot tnh (ch yu l emzyme), protein cu to v protein d tr, trong protein d tr chim t l cao nht. Hm lng protein dao ng t 4,8 n 16,6,% ty vo mi ging. Li dng tnh cht ha tan ca protein trong cc dung mi, ngi ta c th tch trit protein tan t ng phc v cho nhiu mc ch nghin cu nh nh gi cht lng ht, kh nng chu hn Thnh phn ho hc ca ht ng vng u cao hn so vi go trng. Ngoi thnh phn tinh bt, protein, lipid, ng cn cha nhiu loi vitamin, trong vitamin C cao nht. V nhit lng ca ng cao hn go
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trng l 10%. Qua cho thy, ng l cy lng thc c gi tr dinh dng tng i cao (bng 1.1). Bng 1.1. Thnh phn ho hc ca ht ng v go (Phn tch trn 100g) Thnh phn ha hc Go trng Ng vng Tinh bt (g) 65,00 68,20 Protein (g) 8,00 9,60 Lipid (g) 2,50 5,20 Vitamin A (mg) 0 0,03 Vitamin B1 (mg) 0,20 0,28 Vitamin B2 (mg) 0 0,08 Vitamin C (mg) 0 7,70 Nhit lng (Kalo) 340 350 (Cao c im, 1988) [8] 1.1.5. Tnh hnh sn xut ng trn th gii v Vit Nam 1.1.5.1. Tnh hnh sn xut ng trn th gii Trong nhng nm gn y din tch ng trn ton th gii tng ln gp ri, nng sut tng gp 2,5 ln. Din tch ng hng nm khong 139 triu ha, nng sut bnh qun khong 3,8 triu tn/ha, tng sn lng ng trn 525 triu tn/ ha. Ng l cy c a bn phn b vo loi rng nht th gii, tri rng hn 90 v tuyn: T 40 0N ln gn n 55 0B, t cao 1 - 2 m n 400 m so vi mc nc bin [13]. Do , ng c trng hu ht cc ni trn th gii nh Chu M, Chu u, Chu c, Chu Phi. Theo d bo ca Vin nghin cu chng trnh lng thc th gii (IPRI, 2003), vo nm 2020 tng nhu cu ng th gii l 852 triu tn, trong 15% dng lm lng thc, 69% dng lm thc n chn nui, 16% dng lm nguyn liu cho cng nghip. cc nc pht trin ch dng 5% ng lm lng thc nhng cc nc ang pht trin s dng 22% ng lm lng thc (IPRI, 2003) n nm 2020, nhu cu ng th gii tng 45% so vi nhu cu nm 1997, ch yu tng cao cc nc ang pht trin 8

(72%), ring ng Nam nhu cu tng 70% so vi nm 1997 (Bng 1.2), s d nhu cu ng tng mnh l do dn s th gii tng, thu nhp bnh qun u ngi tng, nn nhu cu tht, c, trng, sa tng mnh, dn n i hi lng ng dng cho chn nui tng. Nhng thch thc ln nht l 80% nhu cu ng th gii tng (266 triu tn), li tp trung cc nc ang pht trin. Hn na ch khong 10% sn lng ng t cc nc cng nghip c th xut sang cc nc ang pht trin. V vy, cc nc ang pht trin phi t p ng nhu cu ca mnh (IPRI, 2003). Bng 1.2. D bo nhu cu ng th gii n nm 2020
Vng Nm 1977 (Triu tn) Th gii Cc nc ang pht trin ng Nam Cn Sahara Chu Phi M Latinh Ty v Bc Phi 586 295 136 14 29 75 18 Nm 2020 ( Triu tn) 852 508 252 19 52 118 28 45 72 85 36 79 57 56 % thay i
(Ngun: Vin nghin cu chng trnh lng thc th gii IPRI, 2003) Theo i hc Tng hp Iowa (2006), trong nhng nm gn y khi th gii cnh bo ngun du m ang cn kit, th ng v ang c ch bin ethanol, thay th mt phn nhin liu xng du chy t ti M, Braxin, Trung Quc,... Ring M, nm 2002 - 2003 dng 25,2 triu tn ng ch bin ethanol, nm 2005 - 2006 dng 40,6 triu tn v d kin nm 2012 dng 190,5 triu tn ng (Oxfarm, 2004). Din tch, nng sut, sn lng ng gia cc chu lc trn th gii c s chnh lch tng i ln c th hin bng 1.3
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Bng 1.3. Tnh hnh sn xut ng ca mt s khu vc trn th gii giai on 2005 2007
Din tch Khu vc (Triu ha) Nng sut (T/ha) Sn lng (Triu tn)
2005 Chu u Chu Bc v 11,9 43,7 39,9
2006 15,6 45,0 40,9
2007 2005 2006 2007 13,9 46,4 41,3 46,5 38,3 72,6 61,3 40,7 81,6 49,9 59,1 39,9 75,7 41,7
2005 69,1
2006 96,1
2007 82,6
167,3 183,3 185,4 289,6 333,7 312,0 642,5 724,2 692,0
Trung M Th gii
144,3 146,9
147,0 44,5
(Ngun: S liu thng k ca FAOSTAT, 2008) Qua bng 1.3 cho thy: Din tch trng ng gia cc Chu lc c s chnh lch nhau trong Chu l khu vc c din tch trng ng ln nht, nm 2005 l 43,7 triu ha n nm 2007 l 46,4 triu ha, chim khong 31,6% din tch ng ton th gii. ng v tr th hai l khu vc Bc v Trung M chim khong 28% din tch trng ng th gii. Chu u l khu vc c din tch trng ng thp, chim khong 9,5% din tch trng ng th gii. Nhn chung din tch trng ng ca cc khu vc trn th gii bin ng gia cc nm khng ng k, nu ly 2007 lm mc so snh th Chu u c gim v mt din tch, cn Chu cng vi Bc v Trung M th din tch tng. Bc v Trung M l khu vc c nng sut cao nht t 75,70 t/ha, ng th hai l khu vc Chu u: 59,10 t/ha, v thp nht l Chu 39,91 t/ha (nm 2007). S d Chu c nng sut thp ch yu l do khu vc ny c iu kin thi tit bt thun nh: hn hn, l lt, t canh tc cha thun li. Giai on 2005 2007, Chu v khu vc Bc v Trung M nng sut tng mnh, Chu
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tng 2,4 t/ha, Bc v Trung M tng 9 t/ha, n nm 2007 do nhiu l do khc nhau hai khu vc ny u c nng sut gim. Bc v Trung M l khu vc dn u v sn lng ng trn ton th gii, nm 2005 t 289,6 triu tn, chim 44,45% tng sn lng ng ton th gii. ng th hai l khu vc Chu t 167,3 triu tn, chim 25,87%% tng sn lng ng ton th gii. Nm 2007 khu vc Bc v Trung M t 312,0 triu tn, chim 45,08% tng sn lng ng ton th gii, Khu vc Chu t 185,43 triu tn, chim 26,79% tng sn lng ng ton th gii. Nh vy, trong giai on t nm 2005 n 2007 din tch trng ng trn th gii tng khng ng k, nhng do p dng cc thnh tu khoa hc k thut tin tin c bit l vic m rng din tch trng ng lai nn th gii c s nhy vt v nng sut v sn lng ng, nht l cc nc c nn kinh t pht trin c iu kin thm canh cao v s dng 100% ging ng lai trong sn xut. Trn th gii c mt s nc nh Trung Quc, M, Braxin ch yu l s dng ng lai trong gieo trng v cng l nhng nc c din tch trng ng ln. Tnh hnh sn xut ng ca mt s quc gia trn th gii c th hin qua bng 1.4. Bng 1.4. Tnh hnh sn xut ng ca mt s quc gia trn th gii nm 2007
Tn nc Din tch (Triu ha) Italy M Hy lp Canada Trung Quc n 1,06 30,08 0,84 1,08 26,22 7,40 Nng sut (T/ha) 93,15 100,64 80,95 77,43 50,01 19,60 Sn lng (triu tn) 10,62 280,22 6,80 8,39 131,15 14,50
(Ngun: S liu thng k ca FAO, 2008)
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Qua bng 1.4 cho thy, M l nc c din tch, nng sut, sn lng ln nht t 30,08 triu ha, vi tng sn lng t 280,22 triu tn, nng sut bnh qun t 100,64 t/ha. C th ni, M v Trung Quc l hai cng quc c din tch trng ng ln nht v cao gp nhiu ln so vi cc quc gia khc. Theo s liu ca t chc Nng nghip v Lng thc Lin hp quc (FAO) vic sn xut v tiu th ng trn th gii ang c s mt cn i gia cung v cu dn n tnh trng cc nc nhp khu ng tng dn, cn cc nc xut khu ng th li c xu hng gim. Nc xut khu ng nhiu l M, Trung Quc, Braxin,... cc nc nhp khu ng chnh l Nht Bn, Nam Triu Tin, Malayxia,... i vi cc nc chm pht trin, do iu kin kinh t cn gp nhiu kh khn, u t thp, s dng cc ging ng a phng, ng th phn t do l chnh, cho nn mc d trng vi din tch ln (trn 93,5 triu ha) gp 2 ln cc nc pht trin nhng sn lng li thp hn nhiu, hin nay ch c 20 nc nng sut ng vt mc bnh qun ca th gii. Sn lng ng trn th gii trong nhng nm gn y c chiu hng gim nh, do din tch ng c phn b thu hp nhng sn lng ch gim nhng nc pht trin, cn i vi nhng nc ang pht trin sn lng ng li tng. Nh vy, trn th gii trong nhng nm qua v nng sut ng tng nhanh mt s nc pht trin v cc nc ang pht trin. Hin nay, th trng ng trn th gii c nh gi l mt th trng tng i kh quan. Chnh v vy m sn xut ng trn ton cu s tng trng mnh trong nhng nm ti (theo USDA 1/2003). 1.1.5.2. Tnh hnh sn xut ng Vit Nam Vit Nam, cy ng c trng cch y khong 300 nm v c trng trn nhng iu kin sinh thi khc nhau ca c nc. L cy lng thc quan trng th hai sau cy la, l cy trng chnh pht trin ngnh chn nui. Nng sut ng nc ta trc y rt thp so vi nng sut ng 12

th gii, do s dng ging ng a phng v p dng khoa hc k thut vo sn xut cn hn ch. Phi ti nm 1991 cy ng lai mi bt u c a vo sn xut nc ta, t l trng ging lai t 0,1% nm 1990, nm 2006 tng ln 80% v a Vit Nam tr thnh nc s dng ging lai nhiu v c nng sut cao ca khu vc ng Nam . Bng 1.5. Tnh hnh sn xut ng Vit Nam t nm 2004 n nm 2006 Nm Din tch (1000 ha) 2004 2005 2006 991,10 1052,60 1031,60 Nng sut (T/ha) 34,6171 35,6859 37,024 Sn lng (1000 tn) 343,09 375,63 381,94
nc ta ng c trng hu ht cc a phng c t cao d thot hi nc. Nhng vng trng ng ln l ng Nam B, Ty Nguyn, min ni pha Bc, Trung du ng bng Sng Hng, Duyn hi Min Trung [7] . Trong , khu vc min ni pha Bc trng ch yu l cc ging ng a phng. Nng sut ca cc ging ng a phng thng thp, tuy nhin cc ging ng a phng vn tip tc c quan tm nghin cu v cc u im nh kh nng chu hn, khng su bnh tt v c th gieo trng trn nhiu loi t khc nhau. c bit, nhng nm gn y do k thut canh tc, do mi trng, do s xut hin cc ging ng lai v nhiu nguyn nhn khc dn n s cn kit v ngun gen ging ng a phng. V vy vic su tp, nghin cu v nh gi ngun gen cc ging ng a phng l ht sc cn thit.
1.2. TNH HNH NGHIN CU TNH A DNG DI TRUYN THC VT
1.2.1. Mt s phng php sinh hc phn t s dng trong nghin cu quan h di truyn thc vt
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1.2.1.1. K thut RFLP (Restriction Fragment Length Polymorphisms a hnh di cc on ct gii hn) K thut RFLP l k thut s dng cc endonuclease gii hn ct DNA h gen trnh t nhn bit c trng to ra hng lot on DNA c di xc nh, s lng cc on ny ph thuc vo s im nhn bit trong h gen. a hnh di ny c pht hin bi nh du phng x cc mu d DNA b sung to ra t cng mt locus. RFLP l ch th ng tri. u im ca RFLP l: ch th tin cy trong phn tch lin kt v chn ging v chng c th xc nh c mt tnh trng trng thi ng hp hoc d hp trong mt c th, tn dng c bin d t nhin, pht hin tnh bin d ca DNA trong cc giai on pht trin c quan khc nhau v xy dng quan h di truyn, nghin cu quan h h hng. Nhc im ca RFLP l k thut phc tp, tn km v mt thi gian. Phng php ny i hi mt lng DNA ln (50 200ng t mi c th) [6]. Ignjatovic-Micic D. v cs (2003) s dng k thut RFLP kt hp vi k thut RAPD xc nh quan h di truyn ca 2178 ging ng a phng. Kt qu xc nh khong cch di truyn dao ng t 0,1311 n 0,5075. Tc gi kt lun, d liu phn tch RAPD v RFLP u cho kt qu ging nhau nn c th dng c hai phng php ny xc nh a hnh di truyn [33]. Moretti A v cs (2008) s dng RFLP xc nh quan h di truyn ca Fusarium subglutinans gy bnh ng [37]. 1.2.1.2. K thut AFLP (Amplified Fragment Length Polymorphism a hnh di cc on c nhn bn chn lc) K thut AFLP l k thut kt hp ca RFLP v PCR. AFLP pht hin mt cch c chn lc cc on DNA h gen c ct bi enzyme gii hn v gn vi adaptor (on tip hp). AFLP hot ng da trn nhiu nguyn tc
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tng t nh RAPD, tuy nhin c im khc bit l mi bao gm hai phn: phn c nh di khong 15 bp cha im nhn bit ca enzyme gii hn, phn thay i di khong 2-4 bp. Phn tch sn phm PCR c in di trn gel polyacryamide c phn gii cao. S a hnh c xc nh bi s c mt hay khng c mt ca mt phn on DNA. AFLP rt c hiu qu trong xc nh quan h di truyn v lp bn . Hin nay, phn tch AFLP s dng phng php nhum bc nn d p dng, tuy gi thnh cao hn vi phng php RAPD. K thut AFLP c u im l phn tch a hnh trong khong thi gian ngn, i hi lng DNA t, cho s a hnh cao, tuy nhin vic thit k mi rt phc tp. K thut AFLP c Miranda Oliverira K. v cs, (2004) s dng nh gi mi quan h di truyn ca 96 dng ng lai cng dng, kt qu thu c 638 bng DNA trong c 569 bng DNA a hnh, h s tng ng di truyn dao ng trong khong 0.345 ti 0.891, s quan h di truyn chia 96 dng ng nghin cu thnh 17 nhm [36]. Hartings H. v cs (2008) cng s dng k thut ny xc nh khong cch di truyn ca 54 ging ng Italy [32]. Garcia v cs (2004) kt hp cc k thut RAPD, RFLP, AFLP v SSR nghin cu a dng di truyn ca 18 dng ng lai. S dng k thut AFLP thu c 774 bng a hnh, k thut RAPD khuch i c 262 bng DNA, k thut RFLP thu c 185 bng v SSR nhn c 68 bng a hnh [30]. Thomas Lbberstedt v cs (2000) xc nh quan h di truyn ca mt s ging ng lai bng k thut AFLP vi vic s dng 8 mi AFLP thu c 462 bng DNA a hnh [48]. 1.2.1.3. K thut SSR (Simple Sequence Repeat trnh t lp li n gin) K thut SSR cn c gi l k thut microsatellies (vi v tinh). K thut ny c Litt v Luty pht trin nm 1989 da trn nguyn tc ca PCR [36].
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Trong cu trc h gen ca sinh vt nhn chun tn ti mt lot cc trnh t nucleotide lp li, chng c trng cho loi. SSR gm 2 - 5 nucleotide lp li nhiu ln. V d: (AT)n, (AG)n, (AGTC)n. SSR nm ri rc trong h gen ca thc vt bc cao. on mi c thit k da trn vng bo th hai u ca on SSR. S ln lp li nhiu ln lm cho cc phn on DNA c nhn c di ngn khc nhau. Cc trnh t lp li thng c vng d nhim sc trn mi nhim sc th. Chng c vai tr iu ho hot ng ca cc gen, gp phn lm tng tnh n nh c hc ca nhim sc th trong phn bo, n c th mang thng tin di truyn lin quan n s xc nh gii tnh c ng vt v thc vt. Do s khc nhau v s lng nucleotide trong mi n v lp li m s n v lp li xut hin s a hnh v di ca SSR c nhn bn. Mc a hnh c xc nh sau khi in di sn phm trn gel agarose v gel polyacrylamide. SSR c phn tch trn nh PCR nn ch cn i hi mt lng mu DNA rt nh. SSR l cng c hu ch trong phn tch h gen v chn ging cy trng v y l ch th ng tri c kh nng pht hin tnh a hnh rt cao. Trong thc t ch th ny c s dng nghin cu mt s tnh trng lin quan n nng sut, bnh di, xc nh gii tnh, phn tch quan h di truyn, lp bn genSong nhc im ca ch th ny l s phc tp trong thit k mi v nh gi thit k mi cao. K thut SSR c Qi-lun Yao v cs (2007) s dng xc nh khong cch di truyn ca 54 ging ng t Ty Nam Trung Quc da trn 42 cp mi SSR, tng s alelle thu c l 256, trung bnh mi locus c 6,1 alelle [44]. Legesse B.W. v cs (2007) cng s k thut ny nghin cu mc a dng di truyn v nh gi cu trc gen ca cc dng ng lai cn huyt. Kt qu thu c 104 alelle vi khong cch di truyn xc nh c t 0,28 n 0,73, gi tr PIC l 0,58 [35].
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Sharopova N. v cs (2002) s dng k thut SSR lp bn di truyn i vi cc ging ng nhm phn lp, ch r cc c im ca chng [46]. 1.2.1.4. Bn QTL (Quantiative Trait loci) Bn cc locut tnh trng s lng (QTL) xc nh mi lin kt gia cc phn t vi mt tnh trng hnh thi ang c quan tm. Qua biu QTL c th xc nh c nhng vng nhim sc th c lin quan n mt trng thi. lp bn QTL, cn tin hnh: (1) Xc nh cp lai; (2) Lai v to qun th cho bn ; (3) Theo di s phn ly ca cc ch th trong qun th; (4) X l thng k v lp bn . Lp bn QTL nhm xc nh v tr, hiu qu gen v hot ng ca cc locus lin quan trong tng tc gen v tng tc QTL vi mi trng, t chn lc nh s tr gip ca ch th phn t (MAS-Marker Assisted Selection). K thut QTL cng c Zhu J. v cs (2005) s dng lp bn cho cy ng vi mc ch nhn dng, nh lng trng thi locus, iu ho di ca r ph, xc nh mc mm do ca r nguyn thu, xc nh 6 bn QTL c lin quan ti 53,1% cc bin i phospho ht [53]. 1.2.1. 5. K thut RAPD (Random Amplified Polymorphic DNA) K thut RAPD l k thut phn tch s a hnh cc phn on DNA c nhn bn ngu nhin, do hai nhm nghin cu ca Williams v CS (1990) [52] v Welsh v McClelland (1991) ng thi xy dng [51]. y l mt k thut pht hin ch th di truyn da trn phn ng chui polymerase (PCR) [27]. PCR l mt cng c hu hiu cho vic phn tch h gen thc vt, v n c kh nng to ra mt lng ln cc trnh t DNA c hiu t bt k c th no. Hin nay PCR c xem l phng php nhanh, chnh xc, tng i n gin nh gi sinh vt chuyn gen, phn tch nhanh chng s bin d di truyn phm vi qun th v gia cc c th.
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S hn ch ln nht ca k thut ny l phi da trn mt trnh t DNA c hiu. Tuy nhin, hn ch ny c cc nh sinh hc phn t ci tin, khc phc l k thut PCR s dng on mi ngu nhin hay cn gi l k thut RAPD [34]. Cc thnh phn cn thit tin hnh phn ng RAPD bao gm: - DNA khun (DNA template) vi nng 5 - 50 ng trong 25l. - on mi (primer): Ch s dng mt mi l mt oligonucleotide c trt t nucleotide ngu nhin v c chiu di khong 10 nucleotide. - DNA polymerase: Hot ng ca DNA-polymerase ph thuc vo Mg2+, nng dNTP, pH, nhit bin tnh DNA. DNA-polymerase thng dng l Taq-polymerase. - Bn loi deoxyribonucleotide triphotphat (dNTP). - Ion Mg2+ v dung dch m. Phn ng RAPD c tin hnh qua cc giai on ging nh PCR: - Giai on bin tnh DNA: nhit 950 C trong 30 - 60 giy lm cho hai mch khun tch nhau. - Giai on tip mi: Khi h nhit xung 32 - 400 C, mi tip hp v bm vo si DNA khun. - Giai on tng hp: Nhit c nng ln 72 0 C th cc on mi bt cp vi cc mch n s c ko di vi s tham gia ca Taqpolymerase. Sau mt chu k gm ba giai on nh trn, mt phn on DNA khun c nhn ln thnh hai, cc on DNA c nhn bn trong mi chu k li c coi l DNA khun cho mi chu k nhn bn tip theo. Vy sau k chu k nhn bn s to ra 2k cc on DNA ging on DNA khun ban u. RAPD c th thc hin t 40 - 45 chu k.
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K thut RAPD c u im ch s dng cc mi ngu nhin di 10 nucleotide, qu trnh nhn bn DNA l ngu nhin. on mi ny c th bm vo bt k v tr no c trnh t nucleotide b sung trn phn t DNA h gen. Vi c im l ngn nn xc sut on mi c c im gn trn phn t DNA khun l rt ln. Ty vo nhm, loi thc vt hay vi sinh vt m cc on mi ngu nhin c thit k chuyn dng. Theo l thuyt, s lng cc on DNA c nhn bn ph thuc vo di, v tr ca cc on mi, kch thc v cu trc DNA genome. Thng thng mi on mi ngu nhin s to ra t 2 - 10 sn phm nhn bn. Kt qu l sau khi in di sn phm RAPD s pht hin c s khc nhau trong ph cc phn on DNA c nhn bn. S khc nhau gi l tnh a hnh. Hin tng a hnh cc on DNA c nhn bn ngu nhin xut hin l do c s bin i trnh t nucleotide ti v tr cc on mi lin kt. Sn phm khuch i c phn tch bng in di trn gel agarose hoc polyacrylamide v c th quan st c sau khi gel c nhum bng ho cht c trng. V vy, tnh a hnh thng c nhn ra do s c mt hay vng mt ca mt sn ph m nhn bn [49]. Trong nhng nm gn y, k thut RAPD c s dng rng ri trong nghin cu a dng di truyn. Nhiu nghin cu cho thy, RAPD l mt phng php c hiu qu trong vic xc nh kiu gen, phn tch qun th v ngun gc loi, nghin cu di truyn v lp bn di truyn [3]. Phng php ny cn c ng dng trong vic nh gi b gen ca ging v kh nng phn tch. K thut RAPD cn dng nhn bit, phn loi cc ging cy trng khc nhau nh chui, la m, u , u tng...v pht hin, bo tn s a dng di truyn c bit l cc loi qu him hay mt s ging thc vt a phng. 19

1.2.2. Nghin cu s a dng di truyn thc vt bng k thut RAPD Hin nay, k thut RAPD v ang c ng dng rng ri trong nghin cu v xc nh quan h di truyn thc vt. K thut RAPD cng c ng dng trong vic nh gi a dng di truyn gia cc loi v trong phm vi mt loi phn tch v nh gi h gen thc vt nhm xc nh nhng thay i ca cc dng chn lc mc phn t [40], [41]. Phng php ny cn c ng dng trong vic nh gi b gen ca ging v kh nng phn tch mi quan h di truyn gia cc loi, nhm cc c th cng mt loi [11]. Muthusamy S. v cs (2008) s dng k thut RAPD vi 74 mi ngu nhin v k thut ISSR vi 37 cp mi nghin cu quan h di truyn ca 10 ging u go thu c 987 bng DNA (trong c 719 bng a hnh) t k thut RAPD v 479 bng DNA (trong c 296 bng a hnh) t k thut ISSR, mc a hnh RAPD l 70,3%, mc a hnh ISSR l 60,79% [39]. Afzal v cs (2004) nghin cu tnh a hnh ca tp on ging u xanh nhm chn to ging u xanh c nng sut cao v chu bnh m vng do virus. Cc tc gi s dng 21 ging u xanh vi 34 mi ngu nhin v thu c tng s 204 phn on DNA c nhn bn [23]. Moretzohn v CS nghin cu s a dng di truyn ca lc v mi quan h vi dng di ca chng trn c s phn tch cc vng siu bin ca h gen [38]. nh gi tnh a dng ca mt s ging lc trong tp on ging chng chu bnh g st Yiwu Chen v cs (2006) s dng 11 mi ngu nhin, tc gi nhn c 109 phn on DNA, trong c 66 phn on a hnh, chim 60,6%. iu ny cho thy, trong phm vi ca mi phn ng RAPD gia 33 ging lc nghin cu khc nhau v cu trc DNA, mc sai khc t 4% n 18%. Kt qu phn tch DNA cho thy cc ging lc cng mt vng a l, sinh thi c tp trung thnh tng nhm, gia cc ging chng chu bnh g st ca tp on ging ICRISAT v cc ging nng sut trong 20

nc khng nm trong cng mt nhnh. V th c th la chn cc cp b m mong mun phc v cho cng tc lai ging [28]. Chu Hong Mu v cs (2002) s dng 10 mi ngu nhin so snh h gen ca cc u tng t bin bng k thut RAPD cho thy ba on mi c biu hin a hnh v 6 dng u tng t bin c mc sai khc v b gen [14]. V Th Thng Lan v cs (1999), nghin cu tnh a dng di truyn ca loi rong cu bin cho thy tng s c 46 bng r nt c nhn ngu nhin vi 3 mi (OPA4, PA10, OPL12) v pht hin c s sai khc v mt di truyn ngay trong mt loi rong cu sinh trng trong cc iu kin khc nhau [10]. nh gi s thay i di truyn ca cc dng la ti sinh t m so chu mt nc, inh Th Phng v cs (2001) s dng 10 mi ngu nhin ch ra s sai khc mc phn t gia cc i tng ny [15]. Nguyn Th Tm (2003) s dng 5 mi ngu nhin phn tch tnh a hnh DNA trong b gen ca dng la c chn lc kt qu cc dng c s sai khc mc phn t, trong dng HR128 c h s sai khc vi ging gc l ln nht [16]. Nguyn V Thanh Thanh (2003) nghin cu a dng di truyn ca mt s ging u xanh cho thy trong 5 mi ngu nhin ch c 3 mi RA31, RA45, RA46 cho kt qu a hnh, h s tng ng gia cc ging dao ng t 0,41- 0,80 [17]. K thut RAPD cn l mt cng c rt c hiu qu trong vic tm ra cc ch th phn t phn bit cc ging hay cc loi khc nhau. Raina v cs (2001) s dng ch th RAPD SSR phn tch s a dng h gen v xc nh mi quan h h hng gia cc ging lc trng v lc di [45]. K thut RAPD c s dng rng ri trong nhng nm gn y phn tch di truyn h thng sinh hc. N l phng php hiu qu trong vic
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xc nh kiu gen, phn tch qun th v ngun gc loi, nghin cu di truyn v lp bn di truyn. 1.2.3. Tnh hnh nghin cu s a dng di truyn ca ng bng k thut RAPD Vasconcelos M.J.V.D v cs (2008) s dng k thut RAPD vi 47 mi ngu nhin nhn c 221 bng DNA trong c 130 bng biu hin a hnh [50]. Souza S.G.H.D. v cs (2008) xc nh quan h di truyn ca 16 dng ng lai vi s dng 22 mi RAPD khuch i c 265 bng DNA v 16 cp mi SSR khuch i c 75 bng DNA, 16 dng ng c chia thnh 3 nhm [47]. Okumus A. (2007), 17 ging ng ca Turkey c s dng nghin cu vi 14 mi RAPD thu c 125 bng a hnh (chim 89%), h s sai khc gia cc ging ng t 0,08-0,2 [42]. Abdel - Mawgood A.L. v cs (2006) s dng mt s cc ch th phn t RAPD, SSR, 18S rRNA gen kim tra cc dng ng, trong c ba ging ng t nhin (L1, L2, L3) v hai ging lai F1 bt ngun t h H1 v H2. Hai trong 5 mi RAPD s dng nghin cu biu hin tnh a hnh, mt trong nhng mi cho a hnh xc nh c con lai H2 t hai dng thun. i vi 18S rRNA th khng pht hin c cc bng a hnh [22]. Asif M. v cs (2006) tin hnh phn tch DNA cho 6 ging ng lai bng cch s dng cc mi ngu nhin khuch i a hnh DNA bng k thut RAPD. Tc gi s dng 40 mi ngu nhin, trong c OPR 03, OPR 11 v OPR- 06 cho a hnh. Kt qu kim tra c 3 trong rt nhiu ging ng lai v cng phn bit c ngun gc ca mt s ging ng lai. Tc gi kt lun RAPD l mt cng c hu hiu pht hin s tinh khit ca ging lai nhm nng cao hiu qu trong trng trt v chn nui [25]. 22

Valdemar P. C. v cs (2004) nghin cu khong cch di truyn ca 81 ging ng trong c 79 thuc vng bc Chu u v 2 ging ci tin. S dng k thut RAPD vi 32 mi c khuch i, cao nht l 255 ch th m c 184 bng DNA (chim khong 72,2%) l a hnh. Da trn nhng ch th RAPD, vic lp mt ph h s dng phng php UPGMA. T l kiu di truyn tng t nhau t 0,78 n 0,91. Nhm s liu phn t tp hp thnh 2 nhm chnh, m mu sc ht c tng quan vi nhau [40]. Naureen Z. v cs (2005) nghin cu a hnh ca 30 chng vi khun r ng nhm chn to ging ng cho nng sut cao v tch dng vi khun rhizo r ng. Cc tc gi s dng 30 mi oligonucleotide, kt qu s a dng di truyn t mc ng k vi khong cch di truyn 2 16% [41]. Amorime. P. v cs (2003) s dng cc ch th phn t RAPD v SSR chn lc 13 ging ng ng. Trong , s dng 50 mi RAPD to c 104 bng (chim 72,2%), cn s phn tch SSR nh gi 7 locus vi 42 alen t gi tr PEC t 0,5 n 0,89. c tnh s tng ng di truyn i vi mi RAPD l 0,79, cn ch th SSR l 0,49 [24]. Osipova E.S. v cs (2001) nghin cu s khc nhau di truyn hc gia dng ng A188 v dng soma bt ngun t A188, c nh gi qua s phn tch ca k thut RAPD. S dng 15 mi trong s 17 mi decanucleotie, tng mi cho s khuch i t t 2 17 phn on di 200 2000 bp. M hnh RAPD khng khc gia nhng cc c th ca dng A188, biu hin tnh ng nht v di truyn hc cao. S khc nhau gia dng ban u v dng soma cao t 64 72%. Cn c vo s phn ly di truyn dng soma chia lm 2 nhm. S phn bit nhng nhm dng soma ny ph hp vi ngun gc ca chng [43]. Bauer I (2005) nghin cu c tnh trng thnh sm ca ng lai thu c bng cch nh du protein v RAPD. Khi s dng mi RAPD cho thy t l a hnh cao hn nh du protein [26].
23

Vit Nam, Bi Mnh Cng v cs (2002) tin hnh nghin cu s a hnh di truyn ca mt s ging ng v xc nh c mt s cp lai u t c kh nng cho u th lai cao [5]. Ng Hu Tnh v cs (2002), nghin cu khong cch di truyn ca cc cp lai v nhm u th lai mt ging ng, kt qu tuyn c 7/28 cp lai cho nng sut cao[19]. Ng Vit Anh (2005) s dng k thut RAPD vi 5 mi ngu nhin nhn c 150 phn on DNA c nhn bn ngu nhin t h gen ca 7 ging ng np a phng. C 5 mi s dng trong nghin cu u biu hin tnh a dng v c 16 phn on DNA a hnh chim 51,6% [1]. 1.3. NHN XT CHUNG Trong nhng nm gn y phng hng sn xut ng nc ta l cn tng cng din tch trng ng lai nng xut cao trn c s ng dng cc thnh tu khoa hc k thut nhm tng nng sut t v vt mc trung bnh ca th gii. Tuy nhin, trong thc t sn xut ng Vit Nam vn phi nhp ni rt nhiu cc ging ng m vn c nhng ma b tht thu do ging khng m bo cht lng. V vy, vic nghin cu ha sinh ht ng nhm tm ra cc ging ng c nng sut cao, cht lng tt v n nh l gp phn chn c cc ging c nng sut v cht lng n nh. Nhng cng trnh nh gi s di truyn ca cy ng nc ta cn t. S a dng di truyn s ch ra c mc sai khc gia cc ging ng nghin cu mc phn t ng thi gii thch c tnh a dng ngun gen ca cy ng.
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Chng 2 VT LIU V PHNG PHP
2.1. VT LIU NGHIN CU 2.1.1. Vt liu thc vt S dng 14 ging ng lm vt liu nghin cu. Ngun gc, c im ni thu mu c trnh by bng 2.1. Bng 2.1. Danh sch 14 ging ng nghin cu STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Ging TL VN CB SLO T26 SL SLV TQ L P LC SLT YB T4 Ngun gc Tr Lnh Cao Bng Lu Thng V Nhai Phc Ho Cao Bng Bn Lm Sn La Sng Bng Cao Bng Bn en Sn La Bn Cc Sn La Hm Yn Tuyn Quang Ph Lng Thi Nguyn Ph Bnh Thi Nguyn Than Uyn Lai Chu Bn Cc Sn La Vn Chn Yn Bi Quyt Thng Thi Nguyn c im ni thu mu a hnh cao a hnh cao a hnh cao a hnh cao a hnh cao a hnh cao a hnh cao a hnh cao a hnh cao Trung du a hnh cao a hnh cao a hnh cao Trung du
2.1.2. Ho cht S dng cc loi ho cht tinh khit nhp t cc nc M, Trung Quc, c, Anh, Thy in nh: Tris, EDTA, CTAB, taq polymerase, agarose, buffer PCR, MgCl2, dNTPs, NaCl, sorbitol, NaH2PO4.2H2O v cc ho cht thng dng khc. 25

2.1.3. Thit b Ni hp kh trng (Sturdy - Tai Wan); L vi sng (Sharp); My soi gen tia UV (Weatea - USA); Ngun in di DNA (USA); My li tm (Hettich Germary); My lc nhum DNA (UK); Cn in t; My khuy trn Vortex; My o pH (Mettler Toledo); B n nhit; T sy (Memmert); T cy v trng; T lnh su -20oC, -80oC (Sanyo, Nht Bn); My PCR (Applied Biosystems- M); My quang ph (Thermo Electron). V mt s thit b khc nh: ng eppendorf, u cn, pipet man, ng PCR 2.2. PHNG PHP NGHIN CU 2.2.1. Phng php ha sinh 2.2.1.1 Xc nh hm lng lipid Hm lng lipid c xc nh trn h thng bn t ng Soxhlet ca hng Gerhardt, gm c: bnh cu, tr chit, ng sinh hn. Da vo tnh cht ho tan ca dung mi hu c chit lipid, dung mi hu c c s dng l petroleum ether. 2.2.1.2 Xc nh hm lng protein Hm lng protein c xc nh theo phng php Kjeldahl. Nguyn l: mu c v c ha bng H2SO4 98% kt hp vi cht xc tc chuyn nit hu c thnh (NH4)2SO4 ri dng NaOH y NH3 ra khi mui amoni. NH3 sau khi c gii phng ra s c cun i bng dng hi nc nng. Sau khi c lm ngui s hp th vo dung dch H 3BO3 trong bnh hng to ra mui borat amon c mu xanh trong. xc nh c lng amoniac gii phng ra trong qu trnh chng ct ta em i chun bng dung dch H2SO4 0,1N n khi dung dch chuyn sang mu tm nht. T lng H2SO4 0,1N tiu tn trong qu trnh chun s tnh c lng protein c trong mu. 26

2.2.1.3. Xc nh hm lng ng tan - Xc nh hm lng ng theo phng php Bertrand c m t trong ti liu ca Phm Th Trn Chu v cs [4]. - Nguyn tc: ng trc tip kh oxi ca hydroxit ng mi trng kim mnh lm cho n b kt ta di dng ng ha tr 1 (Cu2O) c mu gch. S lng ng ha tr 1 tng ng vi s lng ng kh theo phng trnh phn ng: RCHO + 2Cu(OH)2 RCOOH + Cu2O + 2H2O Cu+1 + Fe3+ + H2SO4 2CuSO4 + H2O + 2FeSO4 FeSO4 c tnh kh oxi tc dng vi KMnO 4 do dng KMnO4 chun . T s ml KMnO4 0,1N dng chun s xc nh c hm lng ng kh. 2.2.2. Phng php sinh hc phn t 2.2.2.1. Phng php tch DNA t l non ca ng - Quy trnh tch chit v lm sch DNA tng s t l ng theo phng php ca Gawel v cs [31]. Quy trnh tch chit DNA tng s c thc hin theo cc bc sau: - Ly khong 200g l ng non lnh -850C nghin nhanh trong ci chy s c cha nit lng. - B sung 0,8ml m ra, li tm 15 pht, 12000 vng/ pht, loi b dch ni. Bc ny lm 2 ln. - Thm 700l m tch, mix nh, 650C trong 2h, sau ly ra nhit phng 5 pht. - Thm 600l cloroform:isoamyl alcohol (24:1 ), trn u 20 pht - Li tm 15 pht, 12000 vng/pht. Ht cn thn 500l dch trong sang ng eppendorf 1,5 ml mi ( b ta). 27

- Thm 600l isopropanol, lc nh, t ln ( t lnh qua m), ch c dch ta trng. - Li tm 5 pht, 13000 vng/ pht, b dch, p xung giy cho kh. - B sung 300l cn 70%, bng nh. Li tm 5 pht, 13000 vng/ pht, loi b cn (thc hin 2 ln). - Lm kh DNA. - Ha tan DNA trong 50 l nc kh ion. - Kim tra cht lng DNA thu c thng qua in di trn gel agarose 0,8%. - Xc nh hm lng v tinh sch ca DNA trn my quang ph v pha long v nng s dng 10ng/l. 2.2.2.2. Phng php xc nh hm lng v tinh sch DNA tng s * Phng php quang ph hp th Kim tra nng v tinh sch ca DNA trn my quang ph bc sng = 260 nm v = 280 nm. Hm lng v sch ca DNA trong dung dch tch chit c tnh theo cng thc: Hm lng DNA (ng/l) = OD26050 h s pha long. sch DNA = OD260/OD280. Trong : OD260: ch s o c bc sng 260 nm OD280: ch s o c bc sng 280 nm Nu sch DNA = 1,8 - 2,0 th mu c coi l sch. * Phng php in di DNA tng s trn gel agarose - Pha agarose 0,8% trong TAE 1X, un nng cho tan agarose trong l vi sng, ngui khong 60oC; bn gel v i cho kh (khong 1 gi) sau tho lc. - Ly 5 l mu DNA tch c trn vi 2 l dye 6X, tra mu vo ging in di. - Chy in di vi in th 80V trong 30 pht.
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- Ly bn gel nhum ethidium bromide 0,5 l/ml trong 10 pht, ra li bng nc. - Soi bn gel trn my soi gen tia UV, chp nh. 2.2.2.3. Phn ng RAPD Phn ng RAPD c tin hnh vi cc mi ngu nhin theo phng php ca Foolad v cs (1990) [29]. - S dng 10 mi ngu nhin c tng hp ti hng Invitrogen, mi mi di 10 nucleotide, thng tin v trnh t cc mi s dng c trnh by trong bng 2.2 Phn ng RAPD c thc hin trong 20 l dung dch vi thnh phn trong bng 2.3. Tin hnh nhn bn DNA trong my PCR vi chu trnh nhit ca phn ng RAPD l 1 chu k 94oC trong 3 pht; 45 chu k vi nhit (92oC trong 30 giy, 36oC trong 45 giy, 72oC trong 1 pht); 1 chu k 72oC trong 10 pht; lu gi 4oC. Bng 2.2. Trnh t cc nucleotide ca 10 mi RAPD s dng trong nghin cu STT 1 2 3 4 5 6 7 8 9 10 Tn mi M1 M2 M3 M4 M5 M6 M8 M9 RA159 UBC23 Trnh t nucleotide 5 AACCGACGGG 3 5 GGGGGTCGTT 3 5 TACCACCCCG 3 5 GGCGGACTGT 3 5 TCGGCGATAG 3 5 GTGTCTCAGG 3 5 GGAAGTCGCC 3 5 CCTCCAGTGT 3 5 GTCCACACGG 3 5 CCCGCCTTCC 3
29

Bng 2.3. Thnh phn phn ng RAPD STT 1 2 3 4 5 6 7 Thnh phn H2O kh ion Buffer PCR MgCl2 (25 mM) dNTP (2,5 mM) Primer (10 mM) DNA (10 ng/l ) Taq-polymerase Tng
in di sn phm RAPD
Th tch (l) 11,7 2,0 2,0 1,2 1,6 1,0 0,5 20,0
Pha agarose 2% trong TAE 1X. Chy in di vi hiu in th 100V trong 90 pht. Nhum bn gel bng ethidium bromide 0,5 g/ml trong 15 pht, ra sch bng nc, soi gel trn n UV v chp nh.
Phn tch s liu RAPD
Da trn s xut hin hay khng xut hin ca cc phn on DNA khi in di sn phm RAPD ca cc ging ng np vi cc on mi ngu nhin lm c s cho s phn tch s liu theo quy c: S 1: xut hin phn on DNA, s 0: khng xut hin cc phn on DNA. Cc s liu ny c x l trn my vi tnh theo chng trnh NTSYSpc version 2.0 xc nh quan h di truyn ca cc ging ng mc phn t. 2.2.2.4. Phng php x l kt qu v tnh ton s liu Mi th nghim c nhc li 3 ln. S dng ton thng k xc nh tr s thng k nh trung bnh mu ( x ), phng sai (2), lch chun (), v sai s trung bnh mu ( S x ), vi n 30, = 0,05. Cc s liu c x l trn my vi tnh bng chng trnh Excel [21]. 30

Chng 3 KT QU V THO LUN
3.1. C IM HNH THI, HA SINH HT CA CC GING NG NGHIN CU 3.1.1. c im hnh thi ca 14 ging ng nghin cu Hnh thi v khi lng ht l nhng c tnh quan trng trong chn ging ng v n lin quan n cht lng v nng sut. Kt qu nghin cu hnh thi v khi lng 100 ht c trnh by bng 3.1 v hnh 3.1 Bng 3.1. c im hnh thi v khi lng ht ca 14 ging ng np a phng
STT Ging 1 2 3 4 5 6 7 8 9 10 11 12 13 14 TL VN CB SLO T26 SL SLV TQ L P LC SLT YB T4 Hnh thi ht Ht nh, gc cnh Ht trn, my Ht trn, my Ht trn, my Ht dt, my Ht dt, my Ht trn, my Ht trn, my Ht nh, dt, di Ht nh, trn, my Ht trn, my Ht nh, dt, di Ht trn, my Ht nh, dt Khi lng 100 ht (g) 16,09 0,001 16,72 0,002 20,45 0,003 21,21 0,002 28,88 0,003 25,50 0,003 26,81 0,004 23,42 0,001 17,19 0,002 19,63 0,001 24,50 0,001 23,35 0,001 27,42 0,004 25,46 0,003 Mu v ht Trng ng Trng vng, tm Trng ng Trng ng Vng Trng ng Trng c Trng c Trng ng Trng c, tm Trng c Trng c, tm Trng ng Trng ng, vng
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Tnh trng mu sc ht do kiu gen quy nh, t chu nh hng ca mi trng. V bao quanh ht ng l mt mng mng, nhn c mu trng, vng hay tm tu tng ging. Mu sc v ng cng l mt ch tiu quan trng phn loi cc th trong loi ph. Kt qu bng 3.1 cho thy, khi lng 100 ht ca 14 ging ng dao ng trong khong 16,09 g n 28,88 g, cao nht l ging T26, thp nht l ging TL. Khi lng ca ht ph thuc vo kiu gen tng ging. Th t cc ging ng t cao xung thp xp theo khi lng 100 ht l: T26 > YB > SLV > SL > T4> LC > TQ > SLT > SLO > CB > P > L > VN > TL. Tnh trng khi lng ht ph thuc vo kiu gen tng ging. Tuy nhin, khi lng 100 ht c th b thay i nu chu tc ng xu ca mi trng nhng giai on nht nh.
Hnh 3.1. Hnh dng ht ca 14 ging ng K hiu: 1.TL; 2.VN; 3.CB; 4.SLO; 5.T26; 6. SL; 7.SLV; 8.TQ; 9.L; 10.P; 11.LC; 12.SLT; 13.YB; 14.T4. 3.1.2. Hm lng protein, lipid, ng ca 14 ging ng nghin cu nh gi cht lng ht ca 14 ging ng nghin cu, chng ti xc nh hm lng protein, lipid, ng trong ht ca cc ging ng, kt qu phn tch c th hin trong bng 3.2. 32

Bng 3.2. Hm lng protein, lipid, ng trong ht ca 14 ging ng STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Ging TL VN CB SLO T26 SL SLV TQ L P LC SLT YB T4 Protein (%) 9,670,001 10,930,001 11,240,002 10,250,001 11,150,003 10,550,003 12,250,001 11,390,004 10,450,001 11,640,002 7,090,001 8,250,003 8,610,002 7,500,001 Lipid (%) 4,510,01 4,720,06 5,050,05 4,450,02 4,780,01 4,560,04 4,640,05 4,500,06 5,150,02 4,050,01 3,750,05 3,870,04 3,930,03 3,800,03 ng (%) 7,210,01 6,140,02 6,220,02 6,450,05 6,650,03 5,760,03 6,340,04 6,410,01 6,250,02 6,620,04 8,500,05 6,800,01 5,510,03 5,700,01
Bng 3.2 cho thy, hm lng protein ca 14 ging ng dao ng trong khong 7,09-12,25%. Ging SLV c hm lng protein cao nht, cn ging LC c hm lng protein thp nht. Th t cc ging ng t cao xung thp xp theo hm lng protein l: SLV > P > TQ > CB > T26 > VN > SL > OOL > Slo > TL > YB > SLT > T4 > LC. Hm lng protein m chng ti xc nh theo phng php Kendal c thp hn so vi xc nh bng phng php ca Lowry. Ng Vit Anh (2005)
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xc nh hm lng protein tan ca 8 ging ng np theo Lowry, hm lng protein dao ng trong khong 10,5%-14,11% [1]. Hm lng protein trung bnh ca cc ging ng thp hn so vi mt s cy trng nh u tng, u xanh, lc. u xanh, hm lng protein chim khong 24%, u tng chim khong 30-45%, lc khong 26%. Kt qu phn tch hm lng lipid ca cc ging ng cho thy, hm lng lipid trong ht dao ng trong khong 3,75-5,15%. Ging L c hm lng lipid cao nht, cn ging LC c hm lng lipid thp nht. Th t cc ging ng t cao xung thp xp theo hm lng lipid l: L > CB > T26 > VN > SLV > SL > TL > TQ > Slo > P > YB > SLT > T4 > LC.
Hm lng lipid ca ng cao hn u xanh nhng thp hn so vi u tng v lc. Lipid trong ht u tng chim khong 19-25%, u xanh
chim khong 1,3%, lc chim khong 49%. Hm lng lipid lin quan n bo qun ht ging, nhng ging c hm lng lipid cao s kh bo qun. Lipid trong ht ng cha khong 50% acid linoleic, y l acid bo quan trng cn thit cho ngi v ng vt v ng vt khng t tng hp c acid ny. Hm lng ng c xc nh theo phng php Bertrand. Kt qu cho thy, hm lng ng trong ht ca cc ging ng nghin cu cng kh cao, dao ng t 5,51-8,50%. Ging LC c hm lng ng cao nht, cn ging YB c hm lng ng thp nht. Th t cc ging ng t cao xung thp xp theo hm lng ng l: LC > TL > SLT > T26 > P > Slo > TQ > SLV > L > CB > VN > SL > T4 > YB. ng ng vai tr quan trng trong vic iu chnh p sut thm thu trong dch bo khi cy gp iu kin ngoi cnh bt li. Hm lng ng trong ht ca 14 ging ng phn tch cao hn so vi hm lng ng trung bnh ng (3,5%).
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3.2. PHN TCH TNH A HNH DNA BNG K THUT RAPD Cng ngh sinh hc ang c nhiu ng gp c gi tr sn xut nng nghip c bit trong lnh vc chn ging cy trng vi vic s dng cc k thut sinh hc phn t vi mc ch phn tch quan h di truyn v nh gi h gen ca thc vt th RAPD l mt k thut kh thun li v c hiu qu. Trong nghin cu ny, chng ti trnh by kt qu ng dng RAPD vo vic phn tch tnh a hnh DNA ca 14 ging ng np a phng.
3.2.1. Kt qu tch chit DNA tng s t l ng
iu quan tm hng u ca k thut tch chit acid nucleic l thu nhn cc phn t trng thi nguyn vn khng b phn hu bi cc tc nhn c hc hoc b t gy, l iu kin u tin quyt nh cho s thnh cng ca qu trnh nghin cu. Kt qu o ph hp th DNA bc sng 260nm v 280nm c th hin trong bng 3.3. Kt qu cho thy, t s A260/A280 dao ng trong khong 1,8-2,0, chng t DNA tng s thu c m bo cho vic thc hin k thut RAPD. kim tra cht lng DNA tng s, chng ti tin hnh in di trn gel agarose, kt qu c th hin hnh 3.2. 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Hnh 3.2. Hnh nh in di DNA tng s ca 14 ging ng K hiu: 1.TL; 2.VN; 3.CB; 4.SLO; 5.T26; 6.SL; 7.SLV; 8.TQ; 9.L; 10.P; 11.LC; 12.SLT; 13.YB; 14.T4.
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Kt qu kim tra cho thy DNA ca cc mu thu c khng b t gy v sch (hnh 3.2). Khi o OD bc sng 260 nm th ch c mt nh hp th duy nht l 260 nm (hnh 3.3) Bng 3.3. Ph hp th DNA bc sng 260nm v 280nm Tn mu TL VN CB SLO T26 SL SLV TQ L P LC SLT YB T4 A260 0,018 0,015 0,016 0,015 0,018 0,017 0,013 0,014 0,018 0,016 0,015 0,016 0,015 0,017 A280 0,0100 0,0080 0,0084 0,0079 0,0090 0,0090 0,0070 0,0070 0,0090 0,0085 0,0078 0,0081 0,0079 0,0088 A260/A280 1,80 1,88 1,90 1,89 2,00 1,88 1,86 2,00 2,00 1,88 1,92 1,98 1,99 1,93
Nh vy, cc mu DNA thu c u c tinh sch cao, c th s dng cho cc th nghim phn tch DNA tip theo. Sau khi tch chit v tinh sch DNA tng s chng ti tin hnh pha long hm lng DNA v hm lng 10 ng/l phc v cho nghin cu a hnh DNA.
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Hnh 3.3. Ph hp th DNA ca ging SLV o bc sng 260 nm 3.2.2. Kt qu nghin cu a hnh DNA bng k thut RAPD phn tch mi quan h di truyn ca 14 ging ng a phng chng ti s dng 10 mi ngu nhin c di 10 nucleotide vi k hiu M1, M2, M3, M4, M5, M6, M8, M9, RA159, UBC23. Tng s bng xut hin i vi mi mi tng ging ng c thng k v s l kt qu. Kt qu cho thy, trong 10 mi nghin cu th mi M4 xut hin nhiu phn on DNA c nhn bn (13 phn on) vi kch thc quan st t 0,22-1,70kb, nhiu nht l ging LC xut hin 12 bng DNA c nhn bn. Tuy nhin, cng c mt s mi khuch i c rt t bng, l mi M1, M2, M5: Mi M1 nhn bn c mt bng DNA r nt hai ging YB v T4, mi M2 nhn bn c mt bng DNA r nt ging SLV, mi M5 nhn bn c mt bng DNA ging TQ v L (Bng 3.4) Bng 3.4 cho thy, tng s phn on DNA c nhn bn tng ging vi 10 mi ngu nhin dao ng t 41 n 55 phn on, trong ging LC c tng s bng c nhn bn vi 10 mi nhiu nht (55 bng), ging TL c s bng DNA c nhn bn vi 10 mi t nht (41 bng). Tng s phn on DNA c nhn bn ngu nhin thu c t 14 ging ng vi 10 mi l 674.
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Bng 3.4. S phn on DNA xut hin tng ging ng nghin cu
Mi M1 Ging TL VN CB SLO T26 SL SLV TQ L P LC SLT YB T4 Tng 3 2 3 2 3 3 2 3 3 3 3 3 1 1 35 2 2 2 2 2 2 1 3 2 2 3 2 2 2 29 4 5 4 4 3 3 3 4 4 4 4 4 4 5 55 7 8 9 11 8 9 8 10 8 8 12 7 7 11 4 6 4 6 3 6 5 1 1 2 5 5 4 5 8 9 9 9 9 9 9 10 9 10 9 9 10 9 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 2 3 2 2 2 3 2 2 3 3 2 2 2 5 7 9 7 9 8 7 5 4 2 7 7 8 5 90 2 3 4 3 7 7 7 5 6 7 5 5 3 5 69 41 48 51 50 50 53 49 47 43 45 55 48 45 49 674 M2 M3 M4 M5 M6 M8 M9 RA159 UBC23 Tng
123 114 256 112 64
Di y l kt qu phn tch RAPD ca tng mi khi in di sn phm RAPD trn gen agarose 2%. Mi M1 Kt qu in di sn phm RAPD ca 14 ging ng np vi mi M1 c th hin hnh 3.4. Kt qu cho thy, y l mi c s phn on DNA c nhn bn ngu nhin t nht dao ng t 1 n 3 phn on.
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1 M 2 3 4 5 6 7 8 9 10 11 12 13 14
1,4 kb 1,2 kb 0,8 kb
Hnh 3.4. Hnh nh in di sn phm RAPD vi mi M1 ca 14 ging ng K hiu: M: Marker Smart 1.TL; 2.VN; 3.CB; 4.SLO; 5.T26; 6.SL; 7.SLV; 8.TQ; 9.L; 10.P; 11.LC; 12.SLT; 13.YB; 14.T4. Kch thc cc phn on c chiu di c tnh khong 0,7 kb n 1,4 kb. Trong ging TL, CB, T26, SL, TQ, L, P, LC, SLT c s phn on l 3, ging VN, SLO, SLV c 2 phn on, cn ging YB, T4 ch c 1 phn on c nhn bn. Tnh a hnh c th hin s xut hin hay khng xut hin cc phn on DNA c nhn bn ngu nhin khi so snh gia cc ging vi nhau. Ti v tr 1,40 kb ch c ba ging SLT, YB, T4 khng c on DNA c nhn bn, cc ging cn li u xut hin phn on DNA. Ti kch thc 1,20 kb c nm ging VN, SLO, SLV, YB, T4 khng xut hin phn on DNA, cc ging cn li u xut hin phn on. v tr 0,8kb ch c hai ging YB, T4 khng phn on cn li u phn on. kch thc 0,7 kb ba ging SLT, YB, T4 c phn on, cc ging cn li u mt phn on ny. Nh vy, vi mi M1 s phn on DNA c nhn bn 14 ging ng np a phng th hin s sai khc trong cu trc DNA gia cc ging ng np ti bn v tr 1,40 kb, 1,20 kb, 0,8 kb, 0,7 kb. 39

Mi M2 Kt qu in di sn phm RAPD ca 14 ging ng np vi mi M2 c th hin hnh 3.5. Kt qu cho thy, s phn on DNA xut hin khi c nhn bn ngu nhin l 3 phn on. kch thc khong 0,75kb, tt c cc ging u xut hin bng ny. Ch c hai ging TQ v LC xut hin bng DNA kch thc khong 1,2kb. kch thc 1,5kb, ging SLV khng xut hin bng DNA c khuch i, cc ging cn li u xut hin bng ny.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14
2.0 kb 1.5 kb 1.0 kb 0.75 kb 0.5 kb 0.25 kb
Hnh 3.5. Hnh nh in di sn phm RAPD vi mi M2 ca 14 ging ng K hiu: M: Marker 1kb 1.TL; 2.VN; 3.CB; 4.SLO; 5.T26; 6.SL; 7.SLV; 8.TQ; 9.L; 10.P; 11.LC; 12.SLT; 13.YB; 14.T4. Mi M3 Mi M3 khuch i c 5 phn on vi kch thc t 0,5-2,0kb. kch thc 0,7kb, 1,0kb v 1,2kb, tt c cc ging ng nghin cu u xut hin. Mi M4 Kt qu in di sn phm RAPD vi mi RA40 ca 14 ging ng hnh 3.6 cho thy, c t 7 n 12 phn on c kch thc tng ng khong 0,22 kb n 1,7 kb. Ging LC c s phn on DNA c nhn bn nhiu nht 12
40

phn on. Ging SLO, T4 c s phn on DNA c nhn bn 11 phn on. Ba ging TL, SLT, YB c s phn on DNA t nht l 7 phn on. c bit, v tr 0,32kb v 0,80kb ch c ging T4 xut hin phn on DNA c nhn bn, cc ging cn li u khng thy xut hin. kch thc 1,49 kb n 1,70 kb hai ging SLT, YB khng xut hin phn on DNA, cc ging cn li u xut hin. Ti kch thc 0,63 kb ging TL khng xut hin phn on DNA, cc ging cn li u xut hin phn on. DNA c nhn bn kch thc 0,60 kb th cc ging CB, SLO, SL, LC, SLT, YB u xut hin phn on DNA, cc ging cn li mt phn on ny. v tr 0,32 kb ch c ging LC xut hin phn on DNA, cc ging cn li u khng xut hin phn on DNA. Kch thc khong t 0,22 kb n 0,24 kb c cc ging SLO, TQ, LC, T4 u c phn on DNA, 12 ging ng cn li u khng c phn on ny. Ti v tr 1,25 kb, 1,10 kb, 1,0 kb, 0,72 kb, 0,46 kb tt c cc ging u c phn on DNA. Nh vy, vi mi M4 c 13 kch thc (0,22 kb, 0,24 kb, 0,32 kb, 0,60 kb, 0,63kb, 0,80 kb, 1,49 kb, 1,70 kb) th hin tnh a hnh v c 5 kch thc (1,25 kb, 1,10 kb, 1,0 kb, 0,72 kb, 0,46 kb) khng biu hin tnh a hnh.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14
1.5 kb 1.0 kb 0.8 kb
0.6 kb 0.4 kb 0.2 kb
Hnh 3.6. Hnh nh in di sn phm RAPD vi mi M4 ca 14 ging ng K hiu: M: Marker Smart; 1.TL; 2.VN; 3.CB; 4.SLO; 5.T26; 6.SL; 7.SLV; 8.TQ; 9.L; 10.P; 11.LC; 12.SLT; 13.YB; 14.T4. 41

Mi M5 Mi M5 khuch i c 6 phn on vi kch thc t 0,27-1,2kb. tt c cc kch thc xut hin u biu hin tnh a hnh. Mi M6 Mi M6 khuch i c 10 phn on vi kch thc t 0,2-2,0kb. Biu hin a hnh ca cc ging ng nghin cu th hin hai bng kch thc 0,2kb v 0,25kb. 8 kch thc cn li (0,5kb; 0,7kb; 0,9kb; 1,0kb; 1,2kb; 1,4kb; 1,5kb; 2,0kb) khng biu hin a hnh.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14
2.0 kb 1.5 kb 1.0 kb 0.75 kb 0.5 kb 0.25 kb
Hnh 3.7. Hnh nh in di sn phm RAPD vi mi M6 ca 14 ging ng K hiu: M: Marker 1kb 1.TL; 2.VN; 3.CB; 4.SLO; 5.T26; 6.SL; 7.SLV; 8.TQ; 9.L; 10.P; 11.LC; 12.SLT; 13.YB; 14.T4. Mi 8 Mi M8 khuch i c 4 phn on vi kch thc t 0,6-1,5kb. Cc ging ng nghin cu u xut hin cc bng DNA c nhn bn. Nh vy, mi M8 khng biu hin a hnh.
42

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14
1.2 kb 0.9 kb 0.6 kb 0.5 bk
Hnh 3.8. Hnh nh in di sn phm RAPD vi mi M8 ca 14 ging ng K hiu: M: Marker 100bp; 1.TL; 2.VN; 3.CB; 4.SLO; 5.T26; 6.SL; 7.SLV; 8.TQ; 9.L; 10.P; 11.LC; 12.SLT; 13.YB; 14.T4. Mi 9 Hnh 3.9 cho thy, mi M9 khuch i c 3 phn on vi kch thc t 0,4-0,6kb. Ch c kch thc 0,6kb biu hin a hnh, cn kch thc 0,4kb v 0,5kb khng biu hin a hnh (tt c cc ging ng u xut hin 2 bng ny).
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14
0.6 kp 0.5 kb 0.4 kb
Hnh 3.9. Hnh nh in di sn phm RAPD vi mi M9 ca 14 ging ng K hiu: M: Marker 100bp 1.TL; 2.VN; 3.CB; 4.SLO; 5.T26; 6.SL; 7.SLV; 8.TQ; 9.L; 10.P; 11.LC; 12.SLT; 13.YB; 14.T4.
43

Mi RA159 Kt qu phn tch in di sn phm RAPD ca 14 ging ng np vi mi RA159 c th hin qua bng 3.4 v hnh 3.10. Kt qu cho thy, xut hin t 2 n 9 phn on DNA c kch thc tng ng khong 0,21 kb n 1,50 kb. Bn ging VN, CB, T26, SL xut hin phn on DNA v tr 1,50 kb. ba kch thc 0,7 kb, 1,0 kb v 1,20 kb ging P khng xut hin phn on DNA, cc ging cn li u xut hin phn on. Ging VN, CB, T26, LC, SLT, YB xut hin phn on c nhn bn ngu nhin kch thc 0,8 kb. Cn kch thc 0,56 kb ch c ging L khng phn on, cc ging cn li u xut hin phn on. v tr 0,54 kb 6 ging CB, SLO, T26, SL, SLV,YB u phn on DNA, cc ging cn li khng phn on. Ti kch thc 0,36 kb cc ging TL, VN, TQ, L, P, T4 mt phn on. Kch thc 0,21 kb tt c cc ging u phn on DNA.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14
2.0 kb 1.5 kb 1.0 kb 0.8 kb 0.6 kb 0.4 kb 0.2 kb
Hnh 3.10. Hnh nh in di sn phm RAPD vi mi RA159 ca 14 ging ng K hiu: M: Maker Smart 1.TL; 2.VN; 3.CB; 4.SLO; 5.T26; 6.SL; 7.SLV; 8.TQ; 9.L; 10.P; 11.LC; 12.SLT; 13.YB; 14.T4. Nh vy vi mi RA159 c 9 kch thc trong c 8 kch thc (1,5 kb, 1,2 kb, 1,0 kb, 0,8 kb, 0,7 kb, 0,56 b, 0,54 kb, 0,36 kb) th hin tnh a hnh v ch c kch thc 0,21 kb khng biu hin tnh a hnh. 44

Mi UBC23 Phn tch in di sn phm RAPD ca 14 ging ng nghin cu vi mi UBC23 (hnh 3.11) cho thy, xut hin t 2 n 7 phn on DNA c nhn bn ngu nhin. Cc phn on DNA c kch thc c tnh khong t 0,19 kb n 0,64 kb. Trong bn ging T26, SL, SLV, P xut hin 7 phn on DNA v ging TL xut hin phn on DNA thp nht (ch c 2 phn on). Cc ging T26, SLV, L, P xut hin phn on DNA c nhn bn ngu nhin ti kch thc 0,64 kb. T kch thc 0,48 kb n 0,60 kb tt c cc ging u xut hin phn on DNA. kch thc 0,4 cc ging ng nghin cu u xut hin phn on DNA (tr ging TL). Ti v tr 0,38 kb 9 ging ng (TL, VN, CB, SLO,TQ, LC, SLT, YB, T4) mt phn on DNA. Cn v tr 0,36 kb 7 ging ng (T26, SL, SLV, TQ, LC, SLT, T4) u xut hin phn on DNA, cc ging ng cn li mt phn on ny. v tr 0,30 kb cc ging u mt phn on DNA tr ging LC, SLT, T4. Kch thc 0,24 kb ch c hai ging SL, P xut hin phn on DNA c nhn bn, cc ging cn li u khng xut hin phn on. Cc ging u mt phn on DNA ti v tr 0,21 kb tr ging L. Hai ging CB, SL c phn on DNA c nhn bn ngu nhin, cc ging ng cn li u khng phn on DNA v tr 0,20 kb. Ti v tr 0,19 kb cc ging u khng xut hin phn on DNA c nhn bn ngu nhin tr bn ging T26, SLV,TQ, P u xut hin phn on DNA c nhn bn ngu nhin.
45

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14
0.8 kb 0.6 kb 0.4 kb 0.2 kb
Hnh 3.11. Hnh nh in di sn phm RAPD vi mi UBC23 ca 14 ging ng K hiu: M: Marker Smart; 1.TL; 2.VN; 3.CB; 4.SLO; 5.T26; 6.SL; 7.SLV; 8.TQ; 9.L; 10.P; 11.LC; 12.SLT; 13.YB; 14.T4. Nh vy, khi s dng mi UBC23 tnh a hnh biu hin cc kch thc (0,64 kb, 0,4 kb, 0,38 kb, 0,36, 0,3 kb, 0,24 kb, 0,21 kb, 0,2 kb, 0,19 kb), cn 2 kch thc 0,60 kb v 0,48 kb khng biu hin tnh a hnh. T l a hnh ca cc phn on DNA xut hin Vi 140 phn ng PCR c thc hin chng ti in di sn phm v thu c 674 phn on DNA trong 68 loi phn on DNA t 14 ging ng a phng. Kch thc cc phn on DNA c nhn bn trong khong t 0,2kb-2,0kb. S lng cc phn on tng ng vi mi mi nm trong khong 3-13 bng, trong mi M2 v M9 nhn bn c t nht (3 phn on) cn mi M4 nhn c nhiu nht (13 phn on). Qua phn tch in di sn phm RAPD vi 10 mi ngu nhin ca 14 ging ng np a phng nhn thy c 9 mi biu hin a hnh, 1 mi khng biu hin a hnh (mi M8), t l % a hnh c th hin bng 3.5. Bng 3.5 cho thy, mi M1 v mi M5 c s phn on a hnh cao nht (100%) sau l mi RA159 vi t l a hnh chim 88,89%. Mi M8 khng biu hin s a hnh v cc bng DNA nhn bn c u c mt tt c cc mu ng nghin cu. 46

Ng Vit Anh (2005) s dng 5 mi nghin cu a hnh ca 8 ging ng cho thy t l a hnh t t 33,3% n 80% [1]. Nh vy, so vi kt qu th t l phn on a hnh ca 14 ging ng m chng ti nghin cu cao hn. Bng 3.5. T l phn on a hnh khi s dng 10 mi RAPD Mi M1 M2 M3 M4 M5 M6 M8 M9 RA159 UBC23 Phn on DNA c nhn bn 4 3 5 13 6 10 4 3 9 11 Phn on a hnh 4 2 3 8 6 2 0 1 8 9 T l % phn on a hnh 100 66,67 60 61,54 100 20 0 33,33 88,89 81,82
Mi quan h di truyn gia cc ging ng da trn phn tch RAPD Da trn s xut hin hay khng xut hin cc phn on DNA ca cc ging khi in di sn phm RAPD, chng ti xc nh h s a dng di truyn ca cc ging ng np mc phn t. Cc s liu c tnh ton v phn tch theo chng trnh NTSYSpc version 2.0 (Applied Biostatistics Inc., USA., 1998) (theo quy c 1 = xut hin; 0 = khng xut hin). Kt qu nhn c h s tng ng di truyn gia cc ging ng np th hin (bng 3.6)
47

Bng 3.6. H s tng ng di truyn ca 14 ging ng np
Ging TL TL VN CB Slo T26 SL SLV TQ L P LC SLT YB T4 1,00 0,85 1,00 0,84 0,87 1,00 0,84 0,87 0,85 1,00 0,89 0,81 0,85 0,79 1,00 0,79 0,82 0,87 0,87 0,87 1,00 0,78 0,81 0,82 0,82 0,88 0,84 1,00 0,82 0,76 0,75 0,81 0,84 0,77 0,78 0,87 0,81 0,79 0,79 0,85 0,78 0,79 0,81 0,75 0,77 0,74 0,79 0,75 0,79 0,75 0,81 0,82 0,85 0,74 0,78 0,76 0,97 0,82 0,84 0,81 0,78 0,79 0,78 0,75 0,81 0,82 0,82 0,74 0,75 0,77 0,74 0,82 0,72 0,84 0,72 0,74 0,75 1,00 0,84 1,00 0,81 0,85 1,00 0,78 0,71 0,68 1,00 0,74 0,75 0,69 0,84 1,00 0,69 0,71 0,68 0,76 0,90 1,00 0,79 0,75 0,69 0,78 0,82 0,78 1,00 VN CB SLo T26 SL SLV TQ L P LC SLT YB T4
H s tng ng di truyn phn nh quan h di truyn ca cc ging ng vi nhau. Hai ging ng cng gn nhau v mt di truyn th h s tng ng gia chng cng ln v ngc li hai ging c h s tng ng di truyn thp th mi quan h di truyn ca chng cng xa nhau. Bng 3.6 th hin h s tng ng di truyn ca tng cp ging. Kt qu cho thy, h s di truyn ca 14 ging ng dao ng trong khong 0,68 n 0,90. Trong , hai ging SLT v YB c h s ng dng ln nht l 0,90, cn cc cp ging: LC v P, P v YB c h s tng ng nh nht l 0,68. 48

Sau khi phn tch h s ng dng chng ti xy dng s hnh cy (hnh 3.12) ch ra s sai khc di truyn ca cc ging ng. Mc khc nhau c biu hin bng h s sai khc gia cc ging. Cc ging c h s di truyn cao c xp vo mt nhm, gia cc nhm li c lin kt vi nhau.
II
Hnh 3.12. Biu m t quan h di truyn ca 14 ging ng Phn tch hnh 3.12 cho thy, 14 ging ng c chia lm 2 nhm chnh: - Nhm I: gm cc ging TL, L, TQ, P, VN, CB, SLo, SL, T26, SLV - Nhm II: LC, SLT, YB, T4 Hai nhm ng c s sai khc di truyn t 10% n 23% (tc t l tng ng di truyn l 77% n 90%). - Nhm chnh I li chia lm hai nhm ph. Nhm ph th nht gm 4 ging (TL, L, TQ, P); nhm ph th hai gm 6 ging (VN, CB, SL, T26, SLV) vi khong cch di truyn l 20,5% ( 100% - 79,5%).
49

KT LUN V NGH
1. KT LUN 1.1. Khi lng 100 ht ca cc ging ng dao ng t 16,09 g n 28,88 g. Trong , ging T26 c khi lng ht cao nht (28,88g), thp nht l ging TL (16,09g). 1.2. nh gi cht lng ht cho thy, hm lng protein, lipid v ng tng i cao. Hm lng protein trong ht ca 14 ging ng dao ng trong khong 7,09-12,25%, hm lng lipid trong khong 3,75-5,15% , hm lng ng trong khong 5,51-8,50%. 1.3. Bng k thut RAPD vi vic s dng 10 mi ngu nhin nhn c 674 phn on DNA c nhn bn ngu nhin t h gen ca 14 ging ng np a phng. Trong s 10 mi ngu nhin s dng c 9 mi biu hin tnh a hnh. 1.4. H s sai khc di truyn gia cc ging ng dao ng t 10% n 32%. 14 ging ng c chia lm hai nhm chnh vi khong cch di truyn t 10% n 23%.
2. NGH
Kt hp mt s k thut phn tch quan h di truyn khc nh SSR, AFLP, xc nh mi quan h di truyn gia cc ging ng kt qu tin cy hn.
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CNG TRNH CNG B LIN QUAN N LUN VN 1. Nguyn V Thanh Thanh, Trn Th Ngc Dip, Mn Th Hoa, Chu Hong Mu (2009), Nghin cu s a ng di truyn phn t ca mt s ging ng ( Zea mays L.) bng k thut RAPD, Tp ch khoa hc & cng ngh Thi Nguyn, tp 59, s 10, tr 76-80.
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TI LIU THAM KHO
TI LIU TING VIT 1. Ng Vit Anh (2005), Nghin cu c im hnh thi, ha sinh ht, kh nng chu hn v tnh a dng di truyn ca mt s ging ng np a phng, lun vn thc s sinh hc. 2. L c Bin, Nguyn nh Huyn, Cung nh Lng, (1986). C s sinh l thc vt, NXB Nng nghip H Ni. 3. L Trn Bnh v CS, (1997), Cng ngh sinh hc trong ci tin ging cy trng, NXB nng nghip 4. Phm Th Trn Chu, Nguyn Th Hin, Phng Gia Tng (1998), Thc hnh ha sinh, NXB Gio dc 5. Bi Mnh Cng, Trn Hng Uy, Ng Hu Tnh, L Qu Kha, Nguyn Th Thanh (2002), Nghin cu s a dng di truyn ca mt s dng ng ng bng k thut RAPD markers , Tp ch di truyn v ng dng, tr. 16 22. 6. 7. Trnh nh t (2007), Cng ngh sinh hc tp bn, NXB gio dc. Trng Vn ch, (2005), K thut trng ging ng nng sut cao, NXB Nng nghip H Ni, tr. 26. 8. 9. Cao c im (1988). Cy ng, NXB Nng nghip, H Ni. Nguyn Xun Hin v CS, (1972). Mt s kt qu nghin cu v cy ng, NXB khoa hc k thut H Ni. 10. V Thng Lan v cng s (1999), Nghin cu tnh a dng ca mt s li rong cu vng ven bin min nam Vit Nam bng k thut RAPD PCR, Bo co khoa hc hi ngh ton quc, tr. 1321-1327. 11. L nh Lng, Quyn nh Thi (2002), K thut di truyn v ng dng, NXB i hc Quc Gia, H Ni. 52

12. Nguyn c Lng, Dng Vn Sn, Lng Vn Hinh (2000), Gio trnh cy ng, NXB nng nghip. 13. Nguyn c Lng, Dng Vn Sn, Lng Vn Hinh (2002), Gio trnh cy lng thc (dnh cho cao hc), NXB Nng nghip, H Ni. 14. Chu Hong Mu, Nng Th Man, L Xun c, inh Th Phng, L Trn Bnh (2002) nh gi genome ca mt s dng u tng t bin bng k thut phn tch a hnh ca ADN c nhn bn ngu nhin, Tp ch sinh hc 22, tr.21-27. 15. inh Th Phng (2001), Nghin cu kh nng chu hn v chn dng chu hn la bng cng ngh t bo thc vt, Lun n tin s sinh hc, Vin cng ngh sinh hc H Ni. 16. Nguyn Th Tm (2003), Nghin cu kh nng chu cng v chn dng chu nng la bng cng ngh t bo thc vt, Lun n tin s sinh hc, Vin Cng ngh Sinh hc, H Ni. 17. Nguyn V Thanh Thanh (2003), Nghin cu thnh phn ha sinh ht v tnh a dng di truyn ca mt s ging u xanh c kh nng chu hn khc nhau, Lun vn thc s Sinh hc, Trng i hc S Phmi hc Thi Nguyn, tr 48- 67. 18. Ng Hu Tnh (2003), Cy ng. NXB Ngh An. 19. Ng Hu Tnh, Bi Mnh Cng, Ng Minh Tm (2002), Xc nh khong cch di truyn - nhm u th lai - cp lai nng sut cao bng ch th RAPD , Tp ch nng nghip v pht trin nng thn, s 4, tr. 289 - 291. 20. Ng Hu Tnh, Trn Hng Uy, V nh Long, Bi Mnh Cng, L Qu Kha, Nguyn Th Hng, (1997). Cy ng, ngun gc a dng di truyn v pht trin. NXB Nng nghip, H Ni.
53

21. Nguyn Hi Tut, Ng Kim Khi (1996), X l thng k kt qu nghin cu thc nghim trong nng lm ng nghip trn my vi tnh, NXB Nng Nghip, H Ni. TI LIU TING ANH 22. Abdel Mawgood A.L., Ahmed M.M.M, Aliba (2006) Application of molecular markers for hybrid maize (Zea mays L.) identification, International journal of food, agriculture and environment ISSN 14590255 no2, pp. 176-178. 23. Afzal M. A., Muynul Haque M., and Shanmugasumdaram S. (2004), Random Amplified Polymorphic DNA (RAPD) Analyusis of selected mungbean [Vigna radiata (L.) Wilczek] cultivars, Asian Journal of Sciences, 3 (1), pp. 20-24. 24. Amorime. P.; De Souza Almeida C. C.; Melo Sereno M. J. C.; Bered F.; Marbosa J. F., (2003), Genetic variability in sweet corn using molecular markers, Maydia (Maydica) ISSN 1(3), pp. 177-181. 25. Asif M., Rahman M.U.R, Zafar Y., (2006), Genotyping analysis of six maise (Zea mays L.) hybrid using DNA fingerprinting technology pak. J. Bot, 38 (5): 1425 1430. 26. Bauer I., S. Mladenovi Drini, M. Filipovi, Konstanti-nov (2005): Genetic characterization of early maturing maize hybrids (Zea mays L.) obtained by protein and RAPD markers. Genetika, Vol. 37, No. 3, 235-243. 27. Burow M.D., Jesubatham A.M. (2006), PeanutMap: an online genome database for comparative molecular maps of peanut BMC Bioinformatics. 28. Chen Y., Wang D., Arelli P., Ebrahimi M., Nelson R.L., (2006), Molecular marker diversity of SCN-resistant sources in soybean, Genome; 49, 8; ProQuest Central. 0025-6153 Coden Mydcah,
54

29. Foolad, Arusekar, Rodriguer (1995), Application of polymerase Chain Reation (PCR) to plant genome analysis. In: Tissue and organ culture, Fundamenatal methodsSpringer Verlag, Berlin, Heidelerg, 281289. 30. Garcia A.A.F, Benchimol L.L, Barbosa A.M.M, Geraldi I.O, Souza Jr C.L, Souza A.P.D, (2004), Comparison of RAPD, RFLP, AFLP and SSR markers for diversity studies in tropical maize inbred lines, Genet. Mol. Biol., 27 (4). 31. Gawel DNA Jarret, (1991). Genomic DNA Isolation.
http://www.weihenstephan.de/pbpz/bambara/htm/dna.htm. 32. Hartings H, Berardo N, Mazzinelli GF, Valoti P, Verderio A, Motto M., (2008), Assessment of genetic diversity and relationships among maize (Zea mays L.) Italian landraces by morphological traits and AFLP profiling, Theor Appl Genet, 117(6):831-842. 33. Ignjatovie-Micie D., Corie T., Kovacevic D., Markovie K., Lazic-Jancic V., (2003) RFLP and RAPD analysis of maize (Zea mays L.) local populations for identification of variability and duplicate accession, Maydica: 153-159. 34. Innis M. A., Gelfand D. H., Sninsky J. J, White T. J. (1990) PCR protocol: Aguile to methods and ampilication. Academic Press, pp. 482. 35. Legesse BW, Myburg AA, Pixley KV, Botha AM. (2007), Genetic diversity of African maize inbred lines revealed by SSR markers, Hereditas,144(1):10-17 36. Miranda Oliveira K, Rios Laborda P, Augusto F Garcia A, Zagatto Paterniani ME, de Souza AP. (2004), Evaluating genetic relationships between tropical maize inbred lines by means of AFLP profiling, Hereditas. , 140(1):24-33.
55

37. Moretti A, Mul G, Ritieni A, Lday M, Stubnya V, Hornok L, Logrieco A., (2008), Cryptic subspecies and beauvericin production by Fusarium subglutinans from Europe, Int J Food Microbiol. 127(3):312-315 38. Moretzsohn M.C., Hopkins M.S., Mitchell S.E., Kresovich S, Valls J.F., Ferreira M.E. (2004), Genetic diversity of peanut (Arachis hypogaea L.) and its wild relatives based on the analysis of hypervariable regions of the genome, BMC Plant Biol, 14, 4(1). 39. Muthusamy S., Kanagarajan S., Ponnusamy S.(2008), Efficiency of RAPD and ISSR markers system in accessing genetic variation of rice bean (Vigna umbellata) landraces, Electronic Journal of Biotechnology, 11(3). 40. Nanjo T., Kobayashi M., Yoshiba Y., Sanda Y., Wada K., Tsukaya H., Kakubari Y., Yamaguchi Shinozaki K., (2000) Biological funtions of proline in morphogenesis and osmotolerance revealed in antiense transgenic Arabidopsis thaliana http://www.soygenetics.org/
articles/sgu2000-011.htm. 41. Naureen Z., Yasmin S., Hameed S., Malik K.A., Hafeez F.Y. (2005), Characterization and screening of bacteria from rhizosphere of maize grown in Indonesian and Pakistani soils, Journal Basic Microbiol, 45(6): 447-459. 42. Okumus A., (2007), Genetic variation and relationship between Turkish flint maize landraces by RAPD marker, American Journal of Agricultural and Biological Sciences, 2(2): 49-53. 43. Osipova E.S., Koveza O.V., Troitskij A.V., Dolgikh Y.I.,Shamina
Z.B., Gostimskij S.A, (2003), Russian Journal of Genetics, Volume 39, pp. 1412-1419(8).
56

44. Qilun Yao, Kecheng Yang, Guangtang Pan and Tingzhao Rong (2007), Genetic Diversity of Maize (Zea mays L.) Landraces from Southwest China Based on SSR, Genetic Diversity of Maize (Zea mays L.) Landraces from Southwest China Based on SSR, 34(9), 851-860. 45. Raina S.N.V, Kojima T., Ogihara Y., Singh K.P., Devarumath R.M., (2001), RAPD and ISSR figerprints as useful genetic markers for analysis of genetic diversity, varietal identification, and phylogenetic relationships in peanut (Arachis hypogaea L.) cultirs and wild species, Genome, 44(5), 763-72. 46. Sharopova N, McMullen MD, Schultz L, Schroeder S, Sanchez-Villeda H, Gardiner J, Bergstrom D, Houchins K, Melia-Hancock S, Musket T, Duru N, Polacco M, Edwards K, Ruff T, Register JC, Brouwer C, Thompson R, Velasco R, Chin E, Lee M, Woodman-Clikeman W, Long MJ, Liscum E, Cone K, Davis G, Coe EH Jr.(2002) Development and mapping of SSR markers for maize, Plant Mol Biol. 48(5-6):463-481. 47. Souza1 S.G. H. D, Carpentieri P.V, Claudete de Ftima Ruas C. F, Paula C. V, Ruas M. P and Carlos G. A (2008) Comparative Analysis of Genetic Diversity Among the Maize Inbred Lines (Zea mays L.) Obtained by RAPD and SSR Markers, Brazilan archives of biology and tachnology, Vol.51, n. 1 : pp.183-192) 48. Thomas Lbberstedt, Albrecht E. Melchinger, Christina Dule, Marnik Vuylsteke, Martin Kuiper, (2000), Relationships among Early European Maize Inbreds, Crop Science 40:783-791. 49. Valdemar P. Carvalho; Claudete F. Ruas; Josu M. Ferreira ;Rosangela M.P. Moreira; Paulo M. Ruas (2004) Genetic diversity among maize (Zea mays L.) landraces assessed by RAPD markers , Genet. Mol. Biol. Vol.27 No 2 Sao Paulo 1415-4757.
57

50. Vasconcelos M.J.V.D, Antunes M.S., Barbosa S.M., Carvalho C.H.S.D, (2008), RAPD analysis of callus regenerated and seed grownplants of maize (Zea mays L.), Revista brasileira de milho e sorgo, 7(2), pp. 93-104. 51. Welsh J., McClelland M. (1990), "Fingerprinting genomes using PCR with arbitrary primer", Nucl Acids Res,18, pp 7213 - 7218. 52. William J. G. K., Kubelik A. E., Levak K. J., Rafalski J. A., Tingey S. V., (1990), DNA polymorphisms amplified by arbitrary primers are useful as genetic merers, Nucleic Acids Res, 18, pp. 6531-6535. 53. Zhu J., Keappler S.M., Lynch J.P., (2005), Mapping of QTLs for lateral root branching and lengtj in maize (Zea mays L.) under diferentical photphorus supply, Theor Appl Genet, pp. 8-15.
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TRN TH NGC DIP

NGHIN CU TNH A DNG DI TRUYN CA MT S GING NG (ZEA MAYS L.)

lun vn thc s sinh hc

Thi Nguyn: 2009

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TRN TH NGC DIP

NGHIN CU TNH A DNG DI TRUYN CA MT S GING NG (ZEA MAYS L.)

Chuyn ngnh: Di truyn hc M s: 60.42.70

LUN VN THC S SINH HC

Ng-i h-ng dn khoa hc: PGS.TS CHU HONG MU

Thi Nguyn: 2009

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Trn Th Ngc Dip

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Trn Th Ngc Dip

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i ii iii vi vii viii 1 3 3 3 3 5 7 8 8 12

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Bng 1.1 1.2 1.3

Tnh hnh sn xut ng ca mt s quc gia trn th gii nm 2007 11 13 25

1.5 2.1 2.2

2.3 3.1 3.2 3.3 3.4 3.5 3.6

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Hnh nh in di sn phm RAPD vi mi M2 ca 14 ging ng. 40

Hnh nh in di sn phm RAPD vi mi M4 ca 14 ging ng. 41

Hnh nh in di sn phm RAPD vi mi M6 ca 14 ging ng. 42

Hnh nh in di sn phm RAPD vi mi M8 ca 14 ging ng. 43

Hnh nh in di sn phm RAPD vi mi M9 ca 14 ging ng. 43

Hnh nh in di sn phm RAPD vi mi RA159 ca 14 ging ng. 44

Hnh nh in di sn phm RAPD vi mi UBC23 ca 14 ging ng. 46 49

Biu m t quan h di truyn ca 14 ging ng

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