KHOA H p c C 6 N G N G H |
MGHIEIM CLTU D A DAIVG DI TRUYEIXI CLIA 1 6 GIDIXIG O T [Capsicum annuum sppj IXIHAP IXIOI DUA VAO
CHi TH! PHAIXI T I J S S R
Truong Trpng Ngdn', Nguyen Tri Y^n Chi TOMTAT
Su da dang di truyen cua 16 gidng dt nh§p tir AVRDC budc d^u dut^c danh giA bang cdch khao sat 7 mdi cua chi thi phan td SSR. T a t ca cac mdi SSR d^u cho bang da hinh khi di^n di tr^n 16 giong 6t. Trong nghien ciiu nay sd alen dao dong td 3 den 8 alen. Trong 40 alen du<7c p h i t hi^n trung binh c6 5,15 alen trfen m^t locus. Gia tri luong thong tin da hinh (PIC) dao d<)ng tir 0,63 (Hpms 1-69) dtfn 0,85 (Hpms 1-172) va trung binh la 0,76. K^t qua phan tich tr&n gian do hinh cay cho th^y 16 giong tap trung chu y^u vao hai nhom chinh. Hai gidng AVPP0025 va AVPP0414 h^ so tuong ddng 100% v6 mat di tioiy^n. H§ s6 tuong d6ng di truydn cua 16 gidng dao dOng tir 0,68 dt^n 1.00.
Tix khda: Da d^ng di truydn. dt, pbSn tich nhom, PCR SSRs.
L BAT VAIU K
c a y d t d u o c brdng c h u y e u tai c a c tinh m i ^ n T r u n g v a N a m b o , Vi^t N a m . T r o n g n h i i n g n i m g a n day, m d t s d t i n h v i i n g d o n g b a n g s d n g H d n g va d d n g b a n g s d n g C u u L o n g c d n g d a b a t d a u t r o n g d t vdi dien tich n g a y c a n g Idn n h a m c u n g c a p n g u y d n lidu c h o cdc n h a m a y , c a c c d n g t y s a n x u a t c a c m a t h a n g t h u c p h a m d e tieu t h u v a x u a t k h a u , d e m lai loi n h u a n c a o . B e n e a n h d d m d t s d loai d t c d n d u o c d d n g l a m c a y e a n h n h d vao s u d a d a n g vd m a u sSc va d a n g trai ciia n d . Vi v a y d t d a t r d tiianh m d t t r o n g n h i r n g loai c a y t r d n g c d g i a t r i k i n h td c a o v a n h u c a u sir d u n g n g a y c a n g dircfc m d r d n g t r d n k h a p c a n u d e . T u y n h i e n , c d n g t a e g i o n g c h u a d u p e q u a n t a m n h i e u , d d u tij t h a p n d n n S n g s u a t k h d n g c a o . D o d d , vide c a i tao n a n g s u a t v a n a n g c a o c h a t l u o n g d t d a n g l a n h i d m v u h ^ t s u e c a p b a c h m a c a c n h a c h p n g i d n g c a n p h a i q u a n t a m v a t h u c h i e n .
N g h i e n c u u d a d a n g d i t r u y d n l a m o t t r o n g n h i m g b u d c q u a n t r o n g c u a c h u o n g t r i n h c h o n g i d n g cay t r d n g . N g a y n a y , v d i sir tien b d c u a k h o a h o c k y t h u a t , p h u o n g p h a p d a n h g i a d u a v a o d a u p h a n h i A D N d u p e n h i d u n h a k h o a h o e q u a n t a m d o d a u n a y k h d n g c h i u a n h h u d n g b d i giai d o a n s i n h t r u d n g , miia v u v a c a c b i e n p h a p e a n h t a c (Kwon e t al., 2002).
T r o n g s d c a c d a u p h a n tir d a s i t d u n g tiii S S R d u p e s u d u n g r d n g r a i v a h i d u q u a ti-ong viee p h a n tich d a d a n g di t r u y e n , n g h i d n c u u c a u t n i c d i tiny^n q u d n t h e , q u a h i n h tien h o a v a l a p b a n d d g i e n . D a y la d a u
d d n g trdi, cd k h a n&ng p h a t hidn tinh d a h i n h cao v a o n d i n h ( B e e h e r e t al., 2000). M u c tidu c u a n g h i e n c u u n a y n h S m x a c d i n h d u g c d a d a n g di truydn ciia 16 g i d n g d t n h a p npi t h d n g q u a vide k h a o s a t bd gien d u a vao c h i t h i p h a n t u SSR d^ g i u p c d n g t a c c h p n , tao n g u d n vat lieu k h d i d a u p h u c vu c h o c d n g t a c lai tao g i d n g m d i , t h i c h h p p vdi di^u kidn e a n h t a c tai Viet N a m .
L Vk\ UBJ VA PHUONG PHAP NGHIEN CUU 1. v a t h $ u n g h i d n c u u
M u d i s a u g i d n g dt n h S p ndi tir AVRDC n a m 2011 va d a d u p e g i e o d e n g h i d n cuti d a d a n g d i t r u y e n d u a vao c h i t h i p h a n tii S S R D a n h s a c h c u a 16 g i d n g d t d u p e t r i n h b a y d b a n g 1.
B a n g 1. D a n h s a c h c a c g i d n g d t
'Vi?n Nghien cuu & Phat tr'ien Cdng ngh? Sinh hpc, Truong D^ii hpc Can Tho
TT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
T£N GI6NG
A V P P 0 2 0 5 ( G l ) A V P P 9 8 1 3 (G2) A V P P 9 9 0 5 (G3) YI044941 (G4) VI047003 (G5) AVPP0024 (G6) A V P P 0 0 2 5 (G7) AVPP0414 (G8) A V P P 0 5 1 8 (G9) AVPP0609 (GIO) AVPP0610 ( G i l ) A V P P 0 0 2 3 (G12) A V P P 0 2 0 8 (G13) V1046805 (G14) VI037451 (G15) VI037556 (G16)
NGU6N G6C
A V R D C A V R D C A V R D C AVRDC AVRDC A V R D C A V R D C A V R D C A V R D C A V R D C AVRDC A V R D C A V R D C A V R D C A V R D C A V R D C
NONG NGHIPP VA PHAT TRIEN N 6 N G THON - KY 1 - THANG 1/2013 19
KHOA HQC C 6 N 0 WOHt
2. Th6i gian v4 dja d i t o nghien ciru
Thi nghiSm i\i</c thuc hijn Irong n h i ImVi thuOc ViSn Nghidn ciiu vi Phat tri^n C6ng nghf Sinh hpc, truong Dai hpc C4n Tho; thfti gian tu 6/2011 d^n 12/2011.
3. Phuong phip nghidn cihi Bd tri thi nghiem
Thi nghiem dupe bo tri theo kit^ii hoin t o ^ ngau nhitn (Random Complete Dcsign-RCD). M(ii giong dupe gieo trong \<n 3 l4n l5p lai, mfti l5p 1^ lA 1 chau v6i 2 cSy dupe gieo.
Tach chie'tADN
Vide tich chi^t ADN cUrov- thuc hi^n tai Phong Thi nghiem Sinh hpe Phan hi, Vidn Nghidn euu vS
Phat tridn Cong nghd Sinh hpc, truong Dai hpc C ^ Tho. Quy trinh tach chidt dua theo phmmg ph^ip i\(' nghi boi Rogers vi Bendich (1998) v6i vii c-ii tidn.
Mau la tuoi (1 g) di^je l^y liie cSy dat 10-15 ngay tu6i sau khi gieo. La moi giong dupe nghidn trong nita long bang c6i va chay. ADN kdt tua dupe hoa tan
95°C
trong dung dich 10 mM TE v4 ton tru 6 -20°C cho
&in khi sir dyng. NAng dp ADN sau ciing dupe chinh li 20 ng/ml dupe diing cho phin ling PCR.
HhAn mig chudi tiling hfip (PCR) vi ddu SSR PCR dupe thvc hifn trdn miy C1000™Themial Cycler voi did Uch phin ling 25 ^l chiia 0,5 jil dNTPs;
2,5 nl Dream Taq Buffer; \\x\ m6i xu6i; 1 ^l mil ngupc; 0,25 nl BSA, 0,25 \i Taq ADN polymeraza (5 U/iil), 2 \x\ miu ADN h i m lupng 50-100 ng/jU; 17,5 nl nufte c i t 2 Idn.
Sin phfci PCR sau khi khuSch dai dugc phta tich bang didn di gel agaroza 2% trong dung dich ddm TE IX v i chyp bing miy chup hinh gel Biorad UV 20(X). Kdt q u i dupe dpe true Uip trdn gel v4 Uiang chuin 100 bp eiia C6ng ty Fermentas da dugc sii dgng dd u6c lui^ng kieh thu6c doan sin phdm PCR
Chu ky nhidt cua PCR cho cic moi dupe thidt I|p nhu sau:
'
95°C
30" \
\ 50-55°C 30"
72"C
/ ,.
'
72"C
35 chu ky Mdi SSRs
Cac mdi SSRs (Simple Sequence Repeats) dung d6 khao sat bd gien cua cac gidng dt dua vao bao cao ciia Lee et al., 2004. T6n mdi va trinh tir ciia mdi dupe trinh bay d bang 2.
BAng 2. Danh sach 7 mdi (primers) diing trong khio sAt ki^u gien cAc gi6ng dt
Ten Kieu l5p Trinh tu Kich thuCT: (bp)
Hpms 1-1 (CA),saA), Forwars 5' Reverse 5'
tcaacccaatattaaggtcacttcc 3'
ccaggcggggangtagatg 3' 283
Hpms\-Z (AT),o(GT),! Forward 5' Reverse 5'
tgggaaataggalgcgctaaacc 3' aactttaagactcaaaatccataacc 3' Hpms 1-5 (AT)„(GT)„ Forward 5'
Reverses'
ccaaacgaaccgatgaacactc 3' gacaatgttgaaaaaggtggaagac 3' Hpms 1-^9 (AC),(AT), Forward 5'
Reverse 5'
cggtggcatgtagtttctggag 3' aagacatgaaatccacaagttttc 3' Hpmsl-ie& ITA),,(GA).^ Forward 5'
Reverse 5'
gccccgatcaatgaatttcaac 3' tgatttttgggtggagagaaaacc 3' ffpmsl-172 (GA)i5 Forward 5'
Reverse 5'
gggtttgcatgatctaagcatttt 3' cgctggaatgcattgtcaaaga 3' Hpms\-m (GA),6CrG)^ Forwards'
Reverse 5'
tgctgggaaagatctcaaaagg 3' atcaaggaagcaaaccaatgc 3' (Theo Lee etal., 2000).
20 N 6 N G N G H I | P V A PHAT TRIEN N O N G T H O N - KY 1 - THANG 1/2013
KHOA HOC CONG N G H |
Phan tich thdng ke
Phan tich danh gia da dang di tniydn eac gidng duoc su dung he sd tuong dong Jaecard va phuong phap UPGMA h-ong NTSYS 2.1. Ham lu(?ng thdng tin tinh da hinh (PIC - Polymorphic information content) cho mdi locus SSR (i) dupe tinh theo cdng tiiuc (Weir, 1996):
P I C = 1 - I py-
Trong dd: Py la tan sd ciia alen thii i tai locus thirj.
Phan tich nhdm (Cluster analysis)
Viee phan tich nhdm dua vao khoang each di truydn giiia cac gidng bang phuong phap UPCIM.X theo dd nghi cua Nei (1972). Phan mem NTSYSPC v2.1 duoe SIT dung dd vd so dd hinh nhanh (dendrogram).
• . Kir QIMVA THAO LUAN
Ket qua d bang 3 cho thay 7 mdi SSR ddu cho bang da hinh khi dien di tren 16 gidng dt, Trong khi dd Hanacek et al. (2(X)9) da ket luan rang mdi Hpms 1-1, Hpms 1-16S. va Hpms 1-274 khdng cho bang da hinh khi nghien ciiu tinh da hinh ciia ADN U-en 41 gidng dt dd. Trong nghidn ciiu nay sd alen dao dpng tir 3 den 8 alen va trung binh la 5,15 alen tren mdt locus. Sd alen trung binh nay cao hon cac nghien cuu tiudc day la 3,5 alen (Hanacek et al., 2009), 2,9 alen (Minamiyama et al., 2006), 3 alen (Kwon et al., 2007).
Wi\Hpms\-\n
Mdi Hpms 1-3 va Hpms 1-172 cd sd alen cao nhat (8 alien) va mdi cd sd alen thap nh^t (3 alen) la mdi Hpms 1-69. Theo nghidn cuu ciia Lee et al. (2004) mdi Hpms 1-172 cd 9 alen va Hpms 1-69 lai cd 5 alen trdn mgt locus.
Miic dd da dang di truydn kidu gien cua 16 gidng dt nhap ndi dut^e ddnh gia thdng qua gia tri PIC. Gm ti-i PIC dao ddng tir 0.63 (Hpms 1-69) den 0,85 (Hpms 1-172) va trung binh la 0,76 (Bang 3) tuong tu vdi ket qua nghidn cuu cua Lee et al. (2004); gia tri nay cao hon gia tri 0,73 cua HanaCek et al.. 2009 da nghidn cuu.
Bang 3. Kdt qua didn di 7 mdi ciia diu SSR ti-dn 16 gi<^ng6t
Ten moi
Hpms 1-1 Hpms 1-3 Hpms 1-5 Hpms 1-69 Hpms\-V(&
Hpms 1-172 Hpms 1-173 Tong Trung binh
Mhiem sac the 1 9 6 4 16 11 3
Tinh chat
Da hinh Da hinh Da hinh Da hinh Da hinh Da hinh Da hinh
So alen
5 8 5 3 6 8 5 40 5,15
So gidng cho san tham PCR
15 13 14 14 16 16 15 103 14,7
PIC
0,782 0,828 0,786 0,633 0,789 0,859 0,658 5,335 0,762 Moi Hpms 1-69
Hinh 1 Kdt qua didn dl san phSm PCR cua 2 m6i Hpms 1172 v i Hpms 1-69 trdn 16 giong 6t theo thu hi la 1: AVPP0205,2: AVPP9813, 3: AVPP9905,4: VI044941, 5: V1047003,6: AVPP0024,7: AVPP0025,8: AVPP0414,
9: AVPP0518,10: AVPP0609,11: AVPP0610,12: AVPP0023,13: AVPP0208,14: V1046805,15: VI037451 v i 16;
VI037556. MK ladder 100 bp.
Nhdm A gom 5 gidng AVPP0205, AVPP9813, Ket qua xiy dung dupe so do hinh nhinh cua 16
gidng 6t nghidn cihi va duoe trinh bay cr hinh 2. Xet mot cich tong quit vd miic dp phan hi, su da dang di truydn cr so do hinh nhinh cua 16 gidng dt nhip npi da phan lam hai nhdm rd rang v i co 2 gidng AVPP0025, AVPP0414 tuong ddi gidng nhau 100%, do dd khi tidn hanh lai tao khong nen chpn hai gidng niy lai voi nhau. Hd sd tuong quan di huyen bien dpng til 0,68 d^n 1,00.
NONG NGHIEP VA PHAT TRIEN N 6 N G T H 6 N - KY 1 - THANG 1/2013
AVPP9905, V1044941 v i VT047003 cd he sd hiong dong la 0,78 v i sai khac vdi cac gidng thupe nhdm B li 28% (1-0,72).
Nhdm B gdm 11 gidng cdn lai va tiep tuc phan thinh hai nhdm phu v i su sai khae giiia hai nhdm phu la 24,5% (1-0,755). Nhom BI gom cac gidng AVPP0024, AVPP0025, AWP0414, AVPP0518,
KHOA HQC C 6 N G N G H |
AVPP0609 v i AVPP0610 vdi miic dd sai khie di V1037556 vdi miic dO sai khic di truydn dao ddng tir tniydn tir 12%-23%. Nhdm Bll gdm cie gidng 10,5%-24,5%.
AVPP0023, AWP0208, V1046805, V1037451 vi
Hinh 2. So dd hinh nhinh ciia 16 gidng dt nh$p nOi dua vio d^u phin tii SSR vdi thii tv cac gidng GlAVPP0205, G2: AVPP9813, G3AVPP9905, G4: VI044941,05: VI047003, G6: AVPP0024, G7: AVPP0025, G8: AVPP0414, G9: AVPP0518, GIO: AVPP0609, G i l : AVPP0610, 012: AVPP0023, 013: AVPP0208,014:
VI046805, G15: VI037451, v i 016: V1037556.
khang bdnh thin thu ianthracnos^, vi vay chiing la ngudn V9t lidu khoi diu tdt cho chuong trinh cai thi^n gidng sip tdi.
Bay mdi SSR budc dau dupe sii dung cho v i ^ khao sat da dang di tniydn ciia 16 gidng dt nhap npi ddu thd hien kha rd. Miic dp da hinh dao ddng tir 0,63 (Hpms 1-69) ddn 0,85 (Hpms 1-172). Ket q u i phan tich trdn gian dd hinh nhinh cho th^y 16 gidng tap trung chu yd'u vio hai nhdm chinh; nhdm A (5 gidng) va nhdm B (11 gidng). Hai gidng AVPP0025 vi AVPP0414 gidng nhau 100% vd mat di truydn. H^
sd hiong ddng di truydn ciia 16 gidng dao ddng tCr 0,68 ddn 1,00. Su phin nhdm niy sd giup ich nhi^u cho cic nha chon gidng cd hudng chpn cha me tdt dd lai tap nhim cd dupe gidng mdi thich nghi vdi nhidu didu kidn sinh thii khic nhau d ddng being sdng Cuu Long. Cae gidng dt nay da dupe xac dinh l i cd gien
TAI 1(U THAM KHAO
1. Trdn Thj Ba, 2005. Giio trinh hnng rau. Khoa Ndng nghidp, Dai hpc Cin Tha.
2. Beeher, S. A., Steinmetz, K, Weising, K, Boury, S., PelUer, D., 2000. Microsatellites for cultivar identiiieation in Pelargonium. Theor. Appl.
Genet 101,643-651.
3. Haniiek, P., T. Vyhnanek, M. Rohrer, J.
Cieslarovi, H. Stavdlikovi, 2009. DNA polymorphism in genetic resources of red pepper using 22 NONG NGHlfP VA PHAT TRIEN N 6 N G T H 6 N - KY 1 - THANG 1/2013
KHOA HOC C O N G N G H t
microsatellite markers. Hort. Sci. (Prague ), 36, (4): 7. Lee J. M., S. H. Nahm, Y. M. Kim, D. D. Kim, 127-132. 2004. Characterisation and molecular genetic
4. Huang, S., B. Zhang, D. Milboume, L. Cardie, maPPing Pf microsatellite loci in pepper. TheoreUcal 0 . Yang and J. Ouo, 2000. Development of pepper and Applied Genehcs, iOS. 619-627.
SSR markers from sequence databases. Euphytica 8. Minamiyama Y., M. Tsuro, M. Hirai, 2006. An 117:163-167. SSR based linkage map of Capsicum annuum.
5. Kwon, Y. S., Kim, K. M., Kim, D. H., Eun, M. Molecular Breeding, 18.157-169,
Y., and Sohn, J. K, 2002. Marker-assisted 9. Nei, M., 1972. GeneUc distance between introgression of quantitative trait loci associated with populations. Am. Nat. 106: 282-292.
plant regeneration ability in anther eulhire of rice IQ R^^^^^ S. O., and A. Bendich, 1998.
(017-^ saflKsL). Moi. Cells 14. 24-28. g^^^^y^^ „f ^^^^ ^ „ „ , p , ^ ; ^^^^^^ 1„ p i ^ ^ 6. Kwon Y. S., J. Y. Moon, S. I. Yi, K. M. Bae, E. molecular biology manual SecUon A6. Muwer H. Soon, I. H. Cho, B. D. Kim, 2007. CoraparaUve Academic Publisher, Dordreht.pA6/l-A6/ll.
analysis of pepper (^Capsicum annuum L.) vanedes n ^y^j^ g. S., 1996. GeneUc data analysis II, 2"' using morphological characters: AFLP and SSR ^^ Sunderland, Massachusetts, Sinauer Associates:
markers. Korean Journal of Genetics, 29.11-20. gy^
STUDY ON GENETIC POLYMORPHISM OF 16 HOT PEPPER {Capsicum annuum spp.) INTRODUCED VARIETIES BASED ON SSR MARKER
Tniong Trong Ngon, Nguyen Tri Yen Chi Summary
Study on genetic polymorphism of 16 accession of hot pepper {Capsicum annuum spp.) provided by AVRDC was assessed using seven microsatellite markers. All of markers used in this study were polymorphic in 16 pepper vaneties. Three to eight alleles were detected for the remaining loci. In total, 40 alleles were detected, of which, 5.15 alleles per one locus on average. The highest number of different alleles was detected with Hpms 1-3 and Hpms 1-172 primers (8 alleles). Average polymorphism information content (PIC) value was 0.76, ranging from 0.63 (Hpms 1-69) to 0.85 (Hpms 1-172). Cluster analysis of the band patterns separated the varieties into two groups corresponding to varietal types. Two accessions AVPP0025 and AVPP0414 had similarity coefQcient of 1.00. Resistant genes to anthracnose disease were determined in these vaneties, so they can use as parental matenal for crop improvement program.
Keyword: Hot pepper, genetic diversity, cluster analysis, polymerise chian reaction ( PCR), simple sequence repeat (SSRs).
Ngudi phan bi^n: GS.TSKH. Trdn Duy Quy Ng^y nhan bM: 11/10/2012
Ngiy th6ng qua phan bi^n: 24/12/2012 Ngiy duy$t dang: 3/01/2013
NONG NGHlfP VA PHAT TRIEN N 6 N G THON - KY 1 - THANG 1/2013 23
