四川省主要杂交稻亲本的 SSR 多态性分析和指纹图谱的 构建与应用

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1 ( Chinese J Rice Sci), 2006, 20 (1) :1 7 http :/ / www. ricescience. org 1 SSR 1,2,3, # 1,2, # 1,2 1,2, 3 1,2, 3 ( 1, ; 2, ; 3, ; # ; 3, E2mail net) SSR Marker2based Genetic Diver sit y Fingerp rinting of Hybrid Rice in Sichuan, China XIAO Xiao2yu 1,2,3, #, WAN G Yu2ping 1,2, #, ZHAN GJian2yong 1,2, L I Shi2gui 1,2, 3, RON G Ting2zhao 1,2, 3 ( 1 Rice Research I nstitute, S ichuan A g ricultural Universit y, Wenj iang , China; 2 Key L aboratory of Crop Genetic Resources and I m2 provement, Minist ry of Education, S ichuan A gricultural Universit y, Ya an , China; 3 Seed M anagement S tation of S ichuan Prov2 ince, Cheng du , China; # These authors cont y ribute equall y in this paper ; 3 Corres ponding author, E2mail : lishi net) Abstract : A total of 208 SSR primers were used to detect 42 major parents of hybrid rice that were widely popularized in Sichuan Province. Polymorphism were detected with 123 pairs of the primers accounting for % of the total. Fingerprint2 ing maps of these 42 widely used parents were constructed. The results showed significantly higher genetic diversity on chromo2 somes 9 and 10 and considerably lower diversity on chromosome 12. The genetic diversity of these materials was limited, but it was remote correspondingly between the restorer lines and sterile lines. Generally, important parents of hybrid rice can be identified by their particular primers. Out of the primers, 12 and 10 respectively were selected and they were polymorphic be2 tween two parents, which were adapted to examine the purity of hybrid rice seed. The seed purity of D you 527 was identified by the particular SSR primers RM337, RM244 and RM346, and it was agreeable with the result of the field test. Two combi2 nations, D you 527 and D you 68, which rooted in the same sterile line, were distinguished by the particular SSR primer for D you 527. It showed that SSR technique has broad applicability in variety protection, authenticity and purity identification. Key words : hybrid rice ; microsatellite markers ; fingerprinting map ; genetic diversity ; variety identification : DNA , %, 9 10, 12, % 42,,,,SSR DNA,, DNA D 527 RM337 RM244 RM346, D 527, ; D 527 D 68,, : ; ; ; ; : Q943 ; S330 ; S : A : (2006) DNA (DNA fingerp rint) DNA DNA,( ), DNA DNA,; ; ;,,, DNA, ( simple sequence repeat s,ssr), [124 ], SSR DNA [5], 5, : ; : : ( IR T0453) ; (200054) : ( ),,

2 2 ( Chinese J Rice Sci) 20 1 ( ) ; [6 ] 22, 46A 22 ; [7 ] 4 SSR,2A 2B DNA ; [8 ], 6, , ; [9 ] SSR 9, [10 ] [11 ] SSR, PIC SSR RM225 DNA SSR,DNA, DNA, 208 SSR, SSR, D 527, SSR , 18 ( 232A B A A V20A G46A D297A D59A Ap i/ np, Ap i i D702A 7 A A 452 D62A D22A 9B IR6888 D A B),24 ( R R CDR22 IR24 IRBB60 86 R ) D 527, D 527 D ,3,3,, 12, 5, 2002, , D , ; D 527 D 68 36,, 1. 3 SSR 300 D 527, DNA, Murray CTAB [12 ] Cornell SSR, ( 1),527, [13 ] D 527 SSR D 527,D 527 D 68, , 0 1, SPSS 10. 0, ) ( Ap) Ap =, np 2) ( Hs) Hs = 1-2 ( qij / n), qij i j, n 3) PIC Smit h [14 ] PIC (polymorp hism index content ), PIC = 1 - n f 2 i, f ii (allele) PIC SSR, PIC,, 5, ( f = 0. 9),

3 : SSR 3 1 RM Fig. 1. Polymorphism among 42 rice parents as detected wit h SSR marker RM SSR 12 SSR SSR 1, 1 ;,1, 0, DNA 2 Nei D, D = 1 - [2 M xy / ( M x + M y) ], M x M y x y, M xy,spss 10. 0, SSR 208 SSR 123 ( 1 RM339), %,, %,, 12, %( 1) 1 SSR Table 1. Microsatellite markers used in the study. SSR Number of Polymorphism Chromosome Primers of microsatellite primer pairs frequency/ % 1 RM1, RM84, RM86, RM220, RM151, RM259, RM243, RM23, RM81A, RM24, RM140, RM157B,RM129, RM5, RM9, RM34, RM246, RM237, RM128, RM212, RM102, RM265, RM104, RM14, RM35, RM200, RM165, RM238A, RM113, RM259, RM150A, RM100, RM158,RM315,RM283 2 RM211, RM53, RM27, RM29, RM263, RM221, RM240, RM250, RM208, RM48, RM6, RM213,RM207,RM109,RM203,RM251,RM262,RM8,RM154,RM174,RM341,RM166, RM138 3 RM60, RM22, RM231, RM132, RM7, RM218, RM232, RM282, RM338, RM156, RM16, RM49,RM55,RM168,RM293,RM143,RM85,RM36,RM81B,RM227,RM148 4 RM307, RM335, RM261, RM185, RM142, RM119, RM273, RM241, RM303, RM131, RM280,RM226,RM255 5 RM153,RM169,RM161, RM87, RM334, RM164, RM39, RM233B, RM26, RM31, RM249, RM122,RM123,RM163,RM159,RM13,RM173,RM274 6 RM170,RM225, RM253, RM50, RM276, RM136, RM3, RM162, RM30, RM340, RM103, RM204,RM217,RM238B,RM314,RM121,RM275 7 RM125, RM118, RM172, RM214, RM2, RM11, RM10, RM70, RM234, RM18, RM248, RM47,RM51,RM82 8 RM38, RM25, RM44, RM42, RM223, RM210, RM250, RM80, RM230, RM281, RM337, RM152,RM72,RM339,RM264 9 RM219,RM105,RM257,RM107,RM242,RM201,RM215,RM245,RM205,RM RM222, RM244, RM216, RM239, RM184, RM258, RM171, RM147, RM228, RM271, RM304,RM294A,RM RM20B, RM4B, RM167, RM202, RM209, RM229, RM21, RM206, RM254, RM224, RM260,RM332,RM287,RM144,RM120,RM287,RM RM20A,RM4A,RM19, RM247, RM83, RM235, RM17, RM277, RM313, RM309, RM270, RM101,RM260 Total 208

4 4 ( Chinese J Rice Sci) 20 1 ( ) SSR, 42, ( 2), 24,B,, , 527 ;9308 ;IR CDR22 ; 63 R : 6, A 232A V20A G46A A A ; D297A D59A D702A ; 7 A 452 ; D62A D22A ; 9B ; IR6888 D A,, 1 63 F1 63,D22A D62B, ,, SSR,,,,B,,,, ,,,, 21 3, , ( 3),,,, 2. 2 PCR DNA 2 Fig. 2. Dendrogram of similarity among parents of hybrid rice. 3 Fig. 3. Dendrogram of agronomic trait similarity among parents of hybrid rice.

5 : SSR 5 2 DNA SSR Table 2. Character and type of the primers used for DNA fingerprinting. No. Name of t he primer Chromosome Number of allele genes PIC 1 RM RM RM RM RM RM RM RM RM RM RM RM RM RM RM RM RM RM , 18,,26 ( 2), RM PIC, , , SSR 2 5, 52, ,,, DNA,, 1, RM ,, ; RM44 RM D297B, 2 3 RM220 D702A D 527 SSR,D 527 D62A ( 4), RM ; RM337 RM346 D62A,3 D 527 DNA D 527 D 527 RM 244 RM D 527 Fig. 4. Polymorphism of 12 pairs of SSR primers between parents of D you 527. M - Marker (DNA marker DL2000) ; 1,2 - RM6 ; 3,4 - RM17 ; 5,6 - RM55 ; 7,8 - RM211 ; 9,10 - RM224 ; 11,12 - RM337 ; 13,14 - RM164 ; 15,16 - RM163 ; 17,18 - RM153 ; 19,20 - RM341 ; 21,22 - RM346 ; 23,24 - RM244 1,3,5,7,9,11,13,15,17,19,21, ; D62A M, Marker ; Lanes 1 and 2, RM6 ; Lanes 3 and 4, RM17 ; Lanes 5 and 6, RM55 ; Lanes 7 and 8, RM211 ; Lanes 9 and 10, RM224 ; Lanes 11 and 12, RM337 ; Lanes 13 and 14, RM164 ; Lanes 15 and 16, RM163 ; Lanes 17 and 18, RM153 ; Lanes 19 and 20, RM341 ; Lanes 21 and 22, RM346 ; Lanes 23 and 24, RM244 ; Lanes 1,3,5,7,9,11,13,15,17,19,21,23 were Shuhui 527 ; t he ot hers were D62A.

6 6 ( Chinese J Rice Sci) 20 1 ( ) 5 D 527 Fig. 5. Identification of authenticity for D you , RM244 RM346 RM337, D62A D D 68 Every 5 lanes as a group, showing t he amplification result s of primers RM244,RM346 and RM337,lanes 1 to 5 were Shuhui 527, D62A, D you 527, Duoxi 1, and D you 68. RM346 D 527 D 68 PCR, 5 RM244 RM337 DNA, D 527, D62A 527,D 527 D 68, D 527 D 527 RM337 RM244 RM D 527 DNA, ,D62A D 62B %, %,, SSR SSR, SSR,, 3,,,,, [15 ] DNA,, SSR,, SSR, SSR,, SSR,,,,,, 6 RM337 RM244 RM346 D 527 Fig. 6. Amplified result of t he SSR marker RM337, RM244 and RM346 for individuals of D you 527. M - Marker (DNA marker DL2000) ;1 - D62A ; ; D 527 M, DNA marker DL2000 ; Lane 1, D62A ; Lane 2, Shuhui 527 ; Lanes 3 to 29, The individuals of D you 527.

7 : SSR 7,,,,,,,,,,,,,,,,,,, : DNA DNA DNA, DNA,,, 1 DNA,,,,, : 1 Hearne C, Ghosh S, Todd J. Microsatellites for linkage analysis of genetics traits. Trend Genet, 1993, 8 : Akaji H, Yokozeki Y,Inagaki A,et al. Microsatellite DNA mark2 ers for rice chromosome. Theor A p pl Genet, 1996, 93 : ,,,. SSR.,2001,9 (1) : ,,. DNA.,2000,8 (2) : ,,,. DNA.,1999,41 (10) : ,,,. 22.,2000,16 (2) : ,,,. DNA 2A.,2001,15 (3) : SSL P.,2002 (3) : ,,,. SSR.,2003,17 (1) : ,,,. DNA., 2002, 29 (3) : ,,, DNA.,2002,16 (1) : Murray M G, Thompson W F. Rapid isolation of high molecular weight plant DNA. N ucl A ci ds Res, 1980, 8 : Zheng K L, Huang N, Bennett J, et al. PCR2based marker2 assisted selection in rice breeding. IRRI Discussion Paper Series No. 12. Manila : IRRI,1995 : Smit h J S C,Chin E C L,Shu H,et al. An evaluation of t he utili2 ty of SSR loci as molecular markers in maize ( Zea mays L. ) :com2 parisons with data from RFL Ps and pedigree. Theor A p pl Genet, 1997, 95 : ,,,. SSL Ps.,2000,26 (5) :