NGHIN CU A DNG DI TRUYN CC GING LA A PHNG TNH LO CAI BNG CH TH ADN

Trn Danh Su, Nguyn Th Lan Hoa, H Minh Loan, Ng Kim Hoi, Bi Th Thu Giang, Hong Th Hu, H Th Xun Mai, Nguyn Th Tuyt

SUMMARY Evaluation of genetic diversity of local rice (Oryza sativa L.) accessions collected from Lao Cai province at DNA level
The genetic diversity and DNA fingerprinting of 124 local rice accessions collected from three districts of Lao Cai Provinces were elucidated by using 36 simple sequence repeat (SSR) markers distributed through 12 rice chromosomes. The result of SSR fingerprinting showed that total of 235 polymorphic bands were detected at 36 SSR loci. The numbers of polymorphic alleles varied from 3 to 14 depending on each locus, yielding average of 6 alleles per locus. The DNA profiles analysis indicated three out of 36 SSR markers revealed 3 unique alleles (rare alleles) in 3 surveyed rice genotypes. The information of these markers may come in handy to distinctly identify and characterize those 3 local varieties. Cluster analysis based on UPGMA resolved the local rice accessions into two major O. sativa groups, indica and japonica, agreed with classification based on choroplast DNA analysis. This application of DNA polymorphism analysis revealed genomic relationship in Vietnamese rice germplasm, generating a database useful for cultivar identification, local germplasm conservation, and breeding programs. Keywords: Chloroplast DNA, DNA profile, genetic diversity, local rice, SSR markers I. M U Vit Nam l mt trong nhng nc c ti nguyn di truyn la phong ph v a dng trn th gii. Da trn nghin cu v tin ha v s a dng di truyn ca cc loi thuc chi la Oryza, cc nh khoa hc khng nh min Bc Vit Nam nm trong khu vc xut x v a dng di truyn ti a ca loi la trng chu (Oryza sativa) (Chang TT, 1976)[6]. Trong bi cnh nguy c xi mn ngun gen do tc th ha v hin i ha, cho n nm 2009, Trung tm Ti nguyn Thc vt tin hnh thu thp v lu gi hn 2000 ngun gen la ti khu vc thy in Sn La v cc vng ln cn, ni s c di ri dn c tr thnh lng h thy in Sn La. y l vng min ni Ty Bc c cc tiu vng kh hu phong ph v a bn dn c gm nhiu dn tc thiu s v ang sinh sng vi nhng tp qun canh tc v s dng la rt a dng. y l cc ngun gen qu v kh c th tm li c trong tng lai, v vy cn c nh gi chi tit phc v cng tc lu gi v khai thc. Vic nghin cu a dng di truyn, phn loi nhng ngun gen ny l cng tc u tin cn tin hnh. y l cng tc nh gi ban u khng ch c ngha trong vic bo tn cc ging la bn a, m cn c ngha quan trng trong cng tc qun l, khai thc pht trin cc ngun gen c sn. Ngy nay, ch th phn t c s dng rng ri nh mt cng c hu hiu trong nghin cu di truyn v cho php nh gi mt s lng ln locut tri khp b gen ca la. Nhng thng tin v a dng di truyn mc ADN c th pht hin s khc bit nh nht gia cc ging, v th gip nhn dng nhng ging qu, him trong cc tp on ging la trng a phng mt cch nhanh chng v chnh xc. K thut DNA fingerprinting c p dng

nhn dng v phn bit ging hn 30 loi cy trng khc nhau nh cam chanh (Citrus spp), chui (Musa spp.), la m (Triticum spp.), du ty... (Bhat, 2006; Rinehart, 2007)[5] [26]. Trong s cc ch th ADN th ch th SSR c s dng rng ri v hiu qu trong nghin cu cu trc di truyn la trng O. sativa, nghin cu qu trnh tin ha, lm r thun ca vt liu lai to ging... do y l cc ch th ng tri cho a hnh cao v n nh. Hin nay, hn 15.000 ch th SSR c thit lp (www.gramene.org, 2006), ph kn trn bn lin kt di truyn ca la (Giarroccoa v ctv., 2007)[11]. Nhiu nghin cu ng dng SSR v DNA fingerprinting trong nghin cu a dng di truyn nhn dng ging la c cng b trong nhng nm gn y (Kalyan, 2006 [14]; Jalaluddin, 2007 [13]; Muhammad SR, 2009 [19]; Navraj KS, 2009 [20] Trong nghin cu ny, ch th SSR c s dng nghin cu a dng di truyn v lp tiu bn ADN ca 124 ngun gen la thu thp ti 3 huyn ca tnh Lo Cai, nm trong vng lng h thy in Sn La, gip nhn dng ging, pht hin kh nng trng lp, phc v cng tc bo tn ngun gen la bn a. II. VT LIU V PHNG PHP NGHIN CU Vt liu nghin cu - 124 ging la thu thp t vng lng h thy in Sn la v cc vng ph cn: gm 3 huyn Mng Khng, Bc H, Si Ma Cai ca tnh lo Cai ang c lu gi ti Trung tm Ti nguyn thc vt v 2 ging i chng Kasalath, Nipponbare. Cc ging la ny bao gm 68 ging c s ng k chnh thc v c nhn ging ti Hu Lng v Bc t nm 2002 -2009. Cc ging cn li va c thu thp nm 2009. - Ch th ADN lc lp: Cp mi ORF 100 v 50 ch th SSR nh v trn 12 nhim sc th ca b gen la. Phng php nghin cu - ADN l non ca cc ging la nghin cu c tch chit v tinh sch theo phng php CTAB ca Doyle, JJ. v ctv (1987)[8]. - Phn ng PCR vi mi SSR c thc hin vi thnh phn: 1x buffer (Fermentaq), 3mM MgCl2 (Takara), 0,25mM dNTPs, 20mM mi SSR (xui v ngc), 0,5unit Taq (Fermentaq) v 0,25ng ADN tng s la; iu kin nhit: bin tnh 95oC trong 5 pht, 35 chu trnh: 94 oC trong 30 giy, 55 oC trong 40 giy, 72 oC trong 1 pht; tng hp tip 72 oC trong 7 pht, bo qun ti 4 oC. Sn phm PCR c in di trn gel polyacrylamide 8%. - S liu phn tch SSR c x l bng phn mm NTSYS pc2.1 v popgene3.1. H s tng ng di truyn v khong cch di truyn theo cng thc ca Nei (1972) [21]: I=
q12 Q1Q2

v D = - ln (I) D: khong cch di truyn theo cp Q1 v Q2: tng s cc alen ca mu ging 1 v 2

I: H s tng ng di truyn q 12: s cc alen ng nht c 2 mu

S hnh cy c thit lp da trn phng php phn nhm UPGA theo tng ng di (Jaccards/Dice), s dng chng trnh phn mm NTSYS pc2.1. - H s a dng gen (PIC = Polymorphism Information Content) c tnh theo cng thc ca Nei (1973)[22]: PIC = 1 - hk 2 (hk: tn s xut hin ca alen th k). III. KT QU NGHIN CU V THO LUN
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a dng cc ging la nghin cu da trn ch th ADN lc lp Cc ging la trng (Oryza. sativa) Chu bao gm 2 loi ph chnh: Indica v Japonica (Oka, 1988)[24]. Loi ph Indica c xut x nhit i, cn loi ph Japonica bao gm hai loi la c xut x t n i v nhit i. Loi ph Japonica c s a dng di truyn thp hn so vi loi ph Indica (Glaszmann, 1987, Zang v ctv, 1992, Nil. v ctv, 2002)[12], [28], [23]. Trong chn ging, cc php lai xa gia cc loi ph khc nhau thng dn n bt dc v khng hu th th h con lai do ph v cc nhm lin kt v s tng tc gia cc gen c ch trong h gen. Vic gim ti t hp v nhng bin i trong phn ly to ra do lai xa c th gy kh khn trong vic chn lc t hp lai mong mun trong qu trnh chn ging. Do , i vi chn ging, vic xc nh v s dng cc ging b m cho tnh trng mong mun cng nm trong mt phn loi ph c ngha v cng quan trng khc phc hin tng qu khc bit v b gen. Ngoi ra, nhng thng tin v a dng di truyn phn loi cc loi ph s rt c ngha i vi vic xc nh cc gen mc tiu c lp bn . V vy, nhim v phn loi v nghin cu a dng di truyn gia cc loi ph l cn thit, m bo cung cp thng tin cn thit chn cp b m thch hp trong chn ging. C nhiu phng php nh gi phn loi khc nhau: da trn hnh thi, sinh l nh di ca tr gian l mm (mescotyle), s phn hu kim, chiu di/rng ht..., s bt mu phn ng ca ht thc vi dung dch Phenol (Oka, 1958) ,da trn isozyme (Glaszmann (1987), v da trn ch th ADN. Tuy nhin, vic nh gi phn loi di truyn v ngun gc di truyn da trn gen nhn i khi phc tp do c s trao i gen gia la trng v la di khi chng c cng phn b a l (Kuroda v ctv, 2005) [17]. B gen lc lp dng n bi v di truyn theo dng m, trong khi b gen nhn di truyn theo c hai b/m. Vic di truyn n dng ca cc ch th ADN lc lp rt c ngha trong cng tc xc nh mi lin h phn loi di truyn v cng tc xc nh ngun gc la (Kawakami, 2007 [16]. Hn na, la trng c thun ha nh chn lc theo hng c nng sut cao v chng chu tt. Do , nghin cu mi lin h di truyn hay truy tm ngun gc pht sinh ca la, vic la chn v nh gi nhng vng gen khng lin quan n cc c im c chn lc l cn thit. Kanno v ctv. (1993)[15] pht hin mt on ADN khuyt 69 bp ca la Indica so vi la Japonica trong vng ORF100 trn on Pst-12 trong phn t ADN lc lp ca la. on b khuyt ny c pht hin bng k thut PCR v c s dng nh mt ch th phn bit gia loi ph Indica v loi ph Japonica. Cp mi c hiu 5'-ggccatcattttctttag-3' v 5'-agtccactcagccatctctc-3' (k hiu: ORF100) c thit k khuych i on ADN mang vng khuyt thiu 69bp ca vng ORF100 nm trn on Pst-12 ca ADN lc lp (Chen, 1993)[7]. Trong nghin cu ny, 124 ging la bn a c phn loi da trn s khc bit ADN lc lp ca hai loi ph Indica v Japonica bng cp mi ORF100 vi hai ging i chng Nipponbare (la Japonica) v Kasalath (la Indica). Cc ging la nghin cu c gieo v thu l non. Mi mt mu ging, l non ca 10-20 c th c thu gp tch ADN tng s chung. Sau , cc c th ny c tip tc chm sc c th thu mu l tch chit ADN cho cc phn tch c th tip theo. ADN tng s thu c pha long ti cng mt nng v s dng lm khun mu tin hnh phn ng PCR vi cp mi ORF, nhm pht hin a hnh ADN lc lp, ti vng ORF 100, trn on Pst-12. Kt qu phn tch a hnh cho thy c s khc bit r rt gia 2 ging i chng Nipponbare (la Japonica) v Kasalath (la Indica). Ging Kasalath (la Indica) cho sn phm khuych i l bng ADN c kch thc nh hn so vi ging Nipponbare (la Japonica). Trong s 127 ging la nghin cu, 34 ging c kiu ADN lc lp khuyt thiu ti on Pst1 ca
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ORF100 ging Kasalath (Indica), 88 ging cn li u c kiu ADN lc lp khng khuyt thiu ging Nipponbare (Japonica) (Hnh 2) v 5 ging c c hai kiu ADN lc lp. Nh vy, t l la Japonica trong tp on la nghin cu ti Lo Cai chim a s, t 72,13%. Kt qu ny cng ph hp vi cc nghin cu trc y. Trn Danh Su v cng tc vin (2001) [2] s dng k thut RAPD nghin cu cc ging la vng Ty Bc v Ty Nam nc ta cho thy hu ht cc ging la vng Ty Bc thuc loi ph Japonica. Nguyn Th Qunh, (1998) [1] cng pht hin t l Japonica ca la nng vng Ty Bc chim phn ln, trong la rung t l Indica v Japonica gn ngang nhau. Nghin cu phn loi la da trn phn tch ADN nhn v ADN lc lp ca Fukuoka (2001) v a dng di truyn ca 129 ging la bn a ca min Bc Vit Nam bng ch th phn t RFLP v ADN lc lp cng cho thy a phn cc ging la thuc phn loi ph Japonica, tnh trn c la nng v la rung.

Hnh 1: Sn phm PCR ca cc mu ging la nghin cu vi cp mi ORF100

T tri sang phi: 1: 50bp ladder; 2: Kasalath; 3: Nipponbare; 4-30: cc mu ging la nghin cu

Trong , 5 mu ging cho c hai bng ging c 2 ging Kasalath v Nipponbare l cc mu ging c s ng l 1960, 1979, 1982, 4023 v T7013. V th, cc mu ging ny khng th c phn bit bng ch th ORF 100. Mi ORF 100 khuych i a hnh trong on Pst-12 trn vng ORF 100 ca ADN lc lp nm trong t bo cht. ADN lc lp nm trong t bo cht, dng n bi v di truyn theo dng m. Do khng th c hin tng c 2 kiu ADN lc lp trong cng 1 c th. Tuy nhin, trong qu trnh ly mu, 10-20 c th c ct ly l tin hnh tch chit ADN tng s dng cho phn ng, v th, c kh nng c hn mt ging trong mt mu ging ng k. Do vy, chng ti tin hnh tch nhanh ADN tng c th ca cc mu ging ny phn tch, vi s lng mu t 10-15 cc th/mu. y l mu ADN ca cng mt cy c ct l tch chit ADN trong nghin cu phn tch PCR phn loi da trn ADN lc lp trn. Kt qu cho thy trong c 5 mu ging nu trn, tng c th u ch cho mt loi sn phm khuych i ging hoc Kasalath, hoc Nipponbare v khng c c th no mang c hai bng c kch thc khc nhau nh t hp mu ban u. Tuy nhin, tt c cc mu, kt qu u cho hai loi sn phm: c c kiu ADN lc lp ca Kasalath v kiu ADN lc lp ca Nipponbare. iu ny cho thy, cc mu ging ny c s trn ln gia la Indica v la Japonica, da trn phn tch ADN lc lp. y c th l do c im hn ging ca ging a phng. Trong 5 mu ging ny, c 1 mu T7013 va c thu thp nm 2009, cc ging cn li u c nhn ging t cc nm khc nhau.

Hnh 2: Sn phm PCR ca cc c th ca tng mu ging la nghin cu vi cp mi ORF100

A: Cc c th ca mu ging 1960 ;B2: cc c th ca mu ging 4023 nghin cu v xc nh chnh xc ln ging mc ADN nhn, chng ti tin hnh kho st cc mu ging ny vi mt s ch th SSR: RM335, RM340. y l cc ch th c s dng trong cc nghin cu trc y ca B mn a dng sinh hc nng nghip v cho cc sn phm l cc alen c kch thc kh phn bit gia nhm ging Indica v Japonica (Trn Danh Su v cs. 2010) )[4]. ADN ca tng c th trong cc mu ging nghi ln ni trn c s dng lm khun nhn bng phn ng PCR. Sn phm PCR ca tng mu ging c phn tch trn gel polyacrilamid 8% v nhum ethidium bromide. Kt qu phn tch cho thy khng c allen d hp t no c pht hin tng c th trong tng mu ging tuy nhin cc mu ging trn u cho 2 alen.

Hnh 3: Sn phm PCR ca cc cc th ca mu ging s 1960 vi cp mi RM 335 T tri sang phi: DNA ladder, cc ging i chng v ln lt 11 c th ca Acc. 1960 Trong trng hp ca mu ging 1960 vi mi RM 335, 2 allen tch bit c pht hin trong tng s 11 c th nghin cu, 4 c th cho sn phm khuych i l alen c kch thc nh hn, tng ng vi genome ca la Indica, 7 cc th cn li cho alen c kch thc ln hn, tng ng vi la Japonica. Kt qu ny gip cng c gi thuyt ln ging c gii gy ra t l d hp t trong phn tch a hnh ADN lc lp. Lp tiu bn ADN ca cc ging la a phng tnh Lo Cai Trong nghin cu ny, 50 ch th SSR ca la nh v trn 12 nhim sc th c s dng nghin cu a dng di truyn ca cc ging la. ADN tng s c tch chit bng cch trn ln 10-20 c th ca tng mu ging c s dng lm khun mu cho cc phn ng PCR. Tng s 126 mu ging c tin hnh lm PCR, bao gm 2 ging i chng Kasalath v Nipponbare v hai mu ging ln 1979 v 1982 (hai mu ging ny ch ly cc c th cho kiu phn loi Indica). Sn phm PCR c b tr sp xp theo hai nhm la Indica v Japonica theo kt qu phn loi ban u bng ch th ADN lc lp v c in di trn 4 bn gel acrylamide nng 8% vi cng thi gian v in trng. Mi gel c b tr vi DNA ladder X 147 v hai ging la i chng Nipponbare v Kasalath. 34 mu ging Indica v 2 mu ging c tuyn chn nhng c th Indica ca 2 mu ging 1979 v 1982 c load trn bn gel 1.

Hnh 4: Bin ng kch thc ca cc alen ti cc locut SSR kho st Vi 50 ch th SSR, 45 ch th cho sn phm PCR, tuy nhin ch c 36 ch th cho sn phm l cc bng r nt cc mu ging nghin cu, c th s dng lp tiu bn. Thng k trn 36 locut SSR, sn phm PCR l cc bng c kch thc nm trong khong t 80 420 bp (Hnh 4). Tuy nhin, kch thc ph bin ca cc alen thu c trong tp on bin thin trong khong 100- 250. Cc nghin cu v DNA fingerprinting ca la Bangladesh, India v Argentina da trn ch th SSR trc ca Muhammad (2009)[19], Navraj (2009)[20], Rinehart (2007)[26], Kalyan (2006)[14] cng cho kt qu tng t v bin thin kch thc bng ADN. Ti mi locut SSR, kch thc ca alen thu c trong tp on nghin cu bin thin t hn km nhau 1-2 base (RM16, RM172, RM244, RM284, RM346) cho n hng chc, hng trm base (RM164, RM171, RM180, RM335, RM340).

nh gi a dng di truyn cc ngun gen la c ngun gc thu thp thuc khu vc thy in Sn La v cc tnh ln cn bng ch th SSR
nh gi a dng di truyn cc ngun gen la c ngun gc thu thp ti vng lng h thy in Sn La, c th l ti 3 huyn ca tnh Lo Cai, 50 ch th SSR c s dng tin hnh phn tch a hnh di truyn gia cc ngun gen. Trong , 36 ch th cho a hnh ti 1 locut v cho sn phm l cc bng r nt. Ch th RM20 cho a hnh ti hai locut nhng cho sn phm khng r nt v nhiu mu khuyt thiu, nn khng c s dng phn tch a hnh di truyn. Tng s alen c pht hin ti 36 locut l 235. S alen a hnh ti mi locut bin ng t 3 n 14, t trung bnh 6,53/locut. S lng alen nhiu nht l 14 (locut RM335), t 11 alen (locut RM164) v 10 (locut RM21). Locut cho t a hnh nht (3 alen) ti RM172, RM245, RM559. H s a hnh di truyn (PIC) thu c ti cc locut SSR bin ng t 0,33 (locut RM21) n 0,88 (RM335). H s a hnh di truyn (PIC) trung bnh thu c ti cc locut SSR t 0,68, cho thy mc a dng gen tn ti trong 126 mu la nghin cu mc rt a dng. Ch s PIC thp hn cng thu c trong nghin cu a dng la Bangladesh (Jalaluddin, 2007) )[10] (0,44); nghin cu i vi la Japonica ca M (0,46) (Lu, 2005) )[18] v t cao hn so vi h s PIC ca nghin cu a dng la tm (0,35), la np ng bng Bc B (0,43) (Trn Danh Su v cs., 2008, 2010)[3], [4] v nghin cu a dng la temperate Japonica (0,37) (Garris v ctv, 2005)[10]. Trong s 36 locut kho st, 18 locut khng pht hin d hp t, 18 locut cn li pht hin t nht 1 mu ging d hp. Trong , T l alen d hp t quan st c (H) ln nht 2 locut
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RM335 v RM251 (6,35%) , tnh trung bnh t 1,3 % (Bng 3). T l t thp hn so vi t l d hp t ca tp on la np ng bng t 2,4% khi kho st bng 45 ch th SSR trong c 36 ch th tng t trong nghin cu ny (Trn Danh Su v cs. 2010)[4]. Mc d la l loi t th phn, nhng nhiu nghin cu trc cho thy: (i) c hn mt allen ti mt locut ca mt ging v (ii) mc d hp t trung bnh t tng t nh kt qu thu c ca nghin cu ny (Olufowote & ng tc gi, 1997)[25]; Garland & ng tc gi, 1999[9]; Lu & ng tc gi, 2005[18]; Giarrocco & ng tc gi, 2007[11]). Khng ch la, kt qu tng t cng c bo co trong cng trnh nghin cu i vi la mch (Sjakste v ng tc gi, 2002[27]). So snh vi nghin cu a dng di truyn trc trn tp on 45 ging la np ng bng min Bc cho thy, S alen thu c t 36 locut nghin cu kho st ca cc mu ging thu thp t Lo Cai t 235, tng ng vi s alen thu c t 46 locut kho st vi 45 ging la np trong nhin cu trc y, t 234, vi mc alen trung bnh cng t cao hn (so vi 5 alen/locut), nhng t l d hp t li nh hn. S alen a hnh ti mt locut cng bin ng nhiu hn so vi nghin cu trc. cc locut cho a hnh cao (>6) trong nhin cu trc nh RM335, RM164, RM267, RM266, RM21, RM16, RM5, RM251, RM180... chng ti u thu c kt qu a hnh cao hn so vi ln kho st trc trn la np ng bng. Tuy nhin, mc d lng mu ging nhiu hn, nhng ti cc locut RM22 v RM244 s alen a hnh thu c t cc mu ging la ti tnh Lo Cai li t t hn so vi tp on la np bn a ti ng bng sng Hng trc y: ln lt l 7 v 6 so vi 10 v 8. V th, rt c th y l cc locut mang nhiu nt c trng cho la np ca ng bng Sng Hng, v cn c nhiu nghin cu su hn xc nh cc c trng ny. Bng 1: a hnh cc locut SSR cc ging la nghin cu
TT Locut 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 RM5 RM128 RM174 RM263 RM266 RM279 RM509 RM16 RM22 RM251 RM142 RM273 RM335 RM349 RM153 RM164 RM267 RM133 RM340 NST 1 1 2 2 2 2 2 3 3 3 4 4 4 4 5 5 5 6 6 S alen 7 4 5 8 8 7 7 6 7 8 5 6 14 8 5 11 7 4 7 Ging c S alen alen c trng c a (SK) trng PIC
b

H (%) 1,59 0 0 0 4,76 0 0 1,98 0 6,35 5,95 0 6,35 4,37 0 0,4 0,79 0 1,19

TT 20 21 22 23 24 25 26 27 28 29 31 32 33 34 35 36

Locut RM559 RM172 RM180 RM346 RM152 RM264 RM284 RM242 RM245 RM171 RM 216 RM244 RM21 RM332 RM17 RM 19 RM309 Tng s T. bnh

NST 6 7 7 7 8 8 8 9 9 10 10 10 11 11 12 12 12

S alen 3 3 8 4 4 9 4 8 3 4 9 6 10 5 12 4 5 235 6.53

S alen c trng

Ging c alen c trng a (SK)

PIC

H (%) 2,38 0 0 0 1,59 0 0 0 0 0 0,4 1,19 1,19 0 0,79 0,4 5,16 1,3

T7499

1 1

1984 T7022

0.78 0,58 0,48 0,78 0,76 0,77 0,75 0,76 0,77 0,69 0,74 0,68 0,88 0,64 0,66 0,81 0,79 0,65 0,65

0,50 0,59 0,69 0,66 0,37 0,82 0,73 0,84 0,54 0,52 0,75 0,77 0,33 0,70 0,84 0,54 0,64 3 3 0,68

*: PIC: H s a dng gen (PIC = Polymorphism Information Content) c tnh theo cng thc ca Nei (1972) PIC = 1 - p2ij; ( pij l tn sut ca alen th i ti locut th j). **: H: T l d hp t quan st c ti 1 locut

Trong s 36 ch th SSR, ch c 3 ch th cho nhn dng c bit (unique allele) vi 3 alen duy nht ca 3 mu ging T7499, 1984, T7022 (Bng 2). Cc bng c kch thc nm trong khong 100-250bp. Cc nghin cu v DNA fingerprinting ca la Bangladesh, India v Argentina da trn ch th SSR trc ca Muhammad (2009)[19], Navraj (2009)[20], Rinehart (2007)[26], Kalyan (2006)[14] cng cho kt qu tng t v bin thin kch thc bng ADN him.
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Bng 2: Cc ch th SSR cho alen c trng vi cc ging

TT 1 2 3 S ng k 1984 T7022 T7499 Tng s Tn ging Ne Nng Sng C Pl Ma l 3 Kch thc ~ 120 bp ~ 112 bp ~ 145 bp Tn ch th RM335 RM349 RM16 3

B Hnh 5: DNA fingerprinting ca ging la c nhn bit ti cc locut SSR: A: RM335, B: RM349, C: RM16

C Quan h di truyn gia cc ging la v 2 ging i chng c phn tch UPGMA bng phn mm popgene 1.31 v NTSYS 2.1. S hnh cy gia cc ging nghin cu ( s dng phn mm NTSYS 2.1) cho thy h s tng ng di truyn ca c nhm ging bin ng t 0,76 n 0,98 (theo phng php SM, NTSYS). Ti mc tng ng khong 77%, 126 ging la nghin cu phn thnh 2 nhm r rt: Nhm 1 gm Nipponbare v 77 mu ging c kiu ADN khng thiu ca Japonica. Trong nhm 1, cc mu ging la li chia lm hai phn nhm nh: phn nhm1: 51 mu ging la trong a phn l la rung v phn nhm 2 gm 24 ging la trong a phn l cc ging la nng. Nhm 2 gm Kasalath v 47 mu ging, trong gm tt c 34 mu ging c kiu ADN lc lp khuyt thiu ca la Indica. Kt qu cho thy c s tng ng ln gia cch phn loi di loi gia ADN lc lp v genome. Khng c mu ging no c kiu ADN lc lp khuyt thiu ca Indica no c kiu phn nhm thuc Japonica nh a hnh genome ti cc locut SSR. Tuy nhin, mt s mu ging (13 mu ging) c kiu ADN lc lp khng khuyt thiu ca Japonica li c kch thc cc alen tng t cc mu ging la Indica v c phn nhm vi cc ging Indica nh a hnh genome ti cc locut SSR trong nghin cu. H s tng ng di truyn (Nei, 1972) (phn tch bng phn mm PopGene1.31) gia cc ging la nghin cu dao ng t 0,08 n 0,95; thp nht (0,08) gia cp ging T7533 v T7021; tip theo h s tng ng thp cng t c (0,11 v 0,13) l gia mu ging 4024 v ging 4020 vi mu ging T7022 v cao nht (0,95) l gia hai ging 4020 v 4024. Cc ging i chng u c tng ng rt thp so vi cc ging trong nhm la nghin cu: t t 0,05 n 0,45 i vi ging Nipponbare v 0,09 n 0,3 i vi ging Kasalath. Ging kasalath c ngun gc t n v ging Nipponbare c ngun gc t Nht Bn. Cc ging i chng ny tuy u nm trong 2 nhm ln nhng ng tch bit khi cc ging a phng c thu thp ti Lo Cai. Cc cp ging a phn c tng ng di truyn nm trong khong t 0,4-0,6.

Trong cc cp ging, c 2 cp ging c h s tng ng di truyn t 0,9 tr ln: l 4020 v 4024 (0,91), cp 4020 v 4021 (0,95). Hai cp ging ny c h s tng ng ln nht v nn c xem xt trnh loi trng lp da trn cc ch tiu theo di thm v kiu hnh. IV. KT LUN Trong s 36 ch th SSR c s dng trong nghin cu a dng di truyn ca 124 mu ging thu thp, 36 ch th cho a hnh. Trong , nghin cu pht hin 3 locut SSR cho cc bng c trng ca 3 ging la bn a thu thp ti Lo Cai, c th c ng dng trong cng tc to lp b ch th nhn dng ging la bn a v cc ging la Vit Nam ni chung. Cc ging la trong vng thu thp ti Lo Cai rt a dng, tp trung trong 2 phn nhm chnh ca nhm I, gm cc ging la thuc nhm la Japonica. Cc ging ny u mang kiu ADN lc lp khng khuyt thiu ca Japonica. Cc ging la nng v la rung c xu hng nhm vo hai phn nhm trong nhm I. Nhm II gm tt c cc ging la c kiu ADN khuyt thiu ca Indica v 13 ging c kiu ADN lc lp khng khuyt thiu. T l la thuc nhm Japonica t 62% (da trn a hnh ADN lc lp), chim a s trong cc ging la thu thp ti Lo Cai. Trong 124 ging nghin cu, c 2 cp ging c h s tng ng di truyn t 0,9 tr ln: l 4020 v 4024 (0,91), cp 4020 v 4021 (0,95). Hai cp ging ny c h s tng ng ln nht v nn c xem xt trnh loi trng lp da trn cc ch tiu theo di thm v kiu hnh. TI LIU THAM KHO
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