, Waxy : A : (2003)

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1 2003, 26 (3) : 1 6 Journal of Nanjing Agricultural University ( T1 ae stivum L1) Waxy 3, (, ) : SDS2PAGE 293 ( ) Waxy, Wx2A1 2 ; Wx2B1 15, 14 ; Wx2D1 2 ; Wx2A1 Wx2B1 2 ; 3 Waxy, Wx2A1 STS : ; Waxy ; SDS2PAGE; : S51211 : A : (2003) Study on common wheat ( T1 ae stivum L1) for Waxy protein germpla sm YU Chun2mei, CHEN Pei2du 3 (National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agric Univ, Nanjing , China) Abstract : The types of Waxy protein of 293 wheat varieties or lines introduced from various countries were identified by SDS2PAGE method. Two varieties were identified to be the type with null in Wx2A1 protein ; fifteen with null in Wx2B1, among them fourteen were discovered in the present research ; two with null in Wx2D1 ; two with null both Wx2A1 and Wx2B1 subunit and three null in all Wx2pro2 tein. Different null types of Waxy protein were also identified by using PCR2based molecular markers published by other researchers and a new co2dominant STS (special tagged sequence) marker at Wx2A1 locus was developed. Key words : common wheat ; Waxy protein ; SDS2PAGE; molecular marker Wx (granules2bound starch synthase, GBSS ), Waxy, 3 : Wx2A1, Wx2B1 Wx2D1, 7AS 4AL 7DS [1,2 ] Wx2A1a, Wx2B1a Wx2D1a, Wx2A1b, Wx2B1b Wx2D1b [3,4 ] [5 Waxy ], Waxy, [2,5 8 Wx2A1 Wx2B1 Wx2D1, Wx2A1 Wx2B1 ] Nakamura [9 ] Wx2A1 Wx2B1 Wx2D1, 3 Waxy 0, Waxy, Wx2B1 Wx2D1 STS (special tagged sequence), Wx2A1 Wx2D1 SSR (single sequence repeat) [10 12 ] SDS2PAGE 293 Waxy,, Waxy,, : : (1971 ), 3 Corresponding author : (1944 ),, E2mail :

2 , 33 (b) IKE Stur2 dy Satanta ; ; 158/, 158/, 158/, 158/ 107, / , / (a), Eradu/ (a) F [ Waxy SDS2PAGE ],, 5 % SDS 3 5 min, ( r min - 1 ) 5 min, ; 1 ml, 4 1 h, r min min, ;, 4, 40 L, 5 min,, DNA, 2 d,, SDS DNA [14 ] F 1 DNA Wx2A1 STS : 5 2TTGCTGCAGGTAGCCACACCCTG23 [15 ] ; : 5 2GATCCAGCCAGTCAGTAAAG23, [15, 16 ] PCR : 25 L 1 buffer (10 mmol L - 1 Tris2HCl, 50 mmol L - 1 KCl, 115 mmol L - 1 Mg2 Cl 2, ph 810), dntp 800 mol L - 1, 014 mmol L - 1, DNA g, Taq DNA 1 U PCR : 95 5 min ; s, s, s, 35 ; min, 10 : 6 5 L (0125 %, 0125 %, 50 ), 8 % (Acr bis = 39 1), 1 TBE, V, h, EB,, Wx2B1 STS [ 3, 4, 10 ] : 5 2AACCAGCAGCGCTTCAGCCT23, : 5 2 [3 TTGAGCTGCGCGAAGTGCTG23 ] Wx2A1, Wx2D1 SSR [ 4, 11 ] : 5 2CGCTCCCTGAAGAGAGAAAGAA23, [4 : 5 2ATAGGCACAACCCCTAAC23 ], 8 % (Acr bis = ) Waxy SDS2PAGE [13 ] SDS2PAGE Wx2A1 Wx2B1 Wx2D1 Wx2A1 Wx2B1 ( 1 A) Wx2A1 Sturdy HMW ; Wx2B1 Eradu LMW ; Wx2D1 (a) LMW ; Wx2A1 Wx2B1 107 HMW LMW, [6,7 ] (b) Satanta Wx2A1 Wx2B1, IKE 33, [6 ] (b) 33 Wx2D1, Satanta Wx2B1, IKE Wx2A1 Wx2 B1, SDS2PAGE, 293,, Wx2B1 14 : NecRawa AABB/ DD CIMMYTmv CIMMYTmv CIMMYTmv SD3523 SD3367 Manital ( ), NecRawa, Wx2B1 13 SDSPAGE 1 B 1 C 1 B 2, C 6 8, 12, 14 3, (a), (b)

3 3 : ( T1 aestivum L1) Waxy 3 LMW, Wx2B1 NecRawa ( 1 B lane 3) Note : 1) 11Marker ; 21Holland wheat ; 31Synthetic 33 ; 41Neixiang Baihuomai (b) ; 51Jiangsu Baihuomai ; 61Yangmai 158 ; 71IKE; 81 Sturdy ; 91Satanta ; 101Eradu ; 111Kanto 107 ; 121Jiangsu Baihuo2 mai ; 131Neixiang Baihuomai (a) 2) Lane 2, 8 null in Wx2A 1 ; 1 0 null in Wx2B 1 ; 4, 9, 1 1 null in Wx2A1and Wx2B1 ; 5, 12, 13 null in Wx2D1 Note : 1 ) 1 1Marker ; 2 1Mianyang9 924 ; 3 1NecRawa ; 4 1 Eradu ; 5 1 Synthetic AABB / DD ; 6 1 CIMMYTmv ; 7 1 CIMMYTmv ; 81CIMMYTmv ; 91SD3523 ; 101SD3367 ; 111Mian2 yang ; 12. Baihuomai (a) Kanto 107 ; 131Jiangsu Baihuomai ; 141Neixiang 2) Lane 2, 4 11 null in Wx2B1 ; 12 null in Wx2A1and Wx2B1 ; 13, 14 null in Wx2D1 C Note : 1) 11Marker ; 21AR47 (Rye) ; 31Wakanzy (Rye) ; 41 Yangmai 158 ; 51SD3664 ; 61Yangmai ; 71Jiankang 985 ; 81 Yangmai ; 91Waxy wheat 1 ; 101Yangmai No15 ; 111Waxy wheat 2 ; 121Mianyang ; 131Russ ; 141Manital ; 151C1S1 2) Lane 6 8, 12, 14 null in Wx2B1 ; 9, 11 Waxy wheat (null in all Wx protein) 1 Wx SDS2PAGE Fig11 The SDS2PAGE of Wx protein in some wheat analyzed 212 Waxy SDS2PAGE Waxy,,, Wx, Wx2A1 STS2PCR, 2 A N7A/ T7B ( 2 A lane 3), DT4AS N7D/ T7A 593 bp ( 2 A lane 2, 4, 5), 7A 158, 593 bp ; Wx2A1 Stur2 dy, Wx2A1 Wx2B1 107, 574 bp 2 B, 158 ( 2 B lane 7) 107 ( 2 B lane 8) ( 2 B lane 3, 4, 6) F 1, bp ; Wx2 B , Wx2A1, F 1 Wx2A1, Wx2A1 PCR 8 %PAGE bp ( 2 B lane 5), STS2 Fig12 8 % PAGE of PCR product amplified by Wx2A1 spe2 PCR, Wx2A1 cial tagged sequence A Note: 1) 11Marker ( 174, Hind ) ; 21C1S1 ; 31N7A/ T7B ; 41DT4AS; 51N7D/ T7A ; 61Holland wheat ; 71Yangmai 158 ; 81Neixiang Baihuomai ( b) ; 91Sturdy ; 101Satanta ; 111Yangmai 158 ; 121Waxy wheat 1 ; 131Waxy wheat 2 + Yangmai 158 ; 141Waxy wheat 2 ; 151Holland wheat + Yangmai 158 ; 161Yangmai Kanto 107 ; 171Kanto 107 ; 181Yangmai 158 2) Lane 6, 9 null in Wx2A1 ; 8, 10, 17 null in Wx2A1 and Wx2 B1 ; 12, 14 null in all Wx protein B Note : 11Marker ; 21Holland wheat ; 3, 41Yangmai 158/ Waxy wheat 1 ; 51Holland wheat / Yangmai ; 61Yangmai 158/ Waxy wheat 2 ; 71Yangmai 158/ Holland wheat ; 81Yangmai 158/ Kanto 107 ; 91Kanto 107

4 Wx2B1 STS Murai [16 ] Wx, Briney [10 ] STS2PCR, Wx2A1 Wx2B1 Wx2D1 487, bp, , bp 3 ( 3 A lane 2, 3, 8), N7A/ T7B 487 bp ( 3 A lane 4), DT4AS 465 bp ( 3 bp ( 3 A lane 5), N7D/ T7A N7D/ T7B 556 A lane 6, 7) Wx2B1 ( ) ( 3 A lane 9 24, 3 B lane 3 6, 9) 465 bp, SDS2PAGE Vrinten [15 ] Wx2A1b Wx2D1b, Wx2A1b Wx2D1b Wx2A1a Wx2D1a, STS, ( Wx2A1b Wx2D1b) Wx2A1 Wx2D1 ( Wx2A1a Wx2D1a) 487 bp 556 bp, STS Wx2A1 Wx2B1 Satanta ( 3 A lane 15) 107 ( 3 B lane 4) 3 3 Wx2B1 PCR 8 %PAGE waxy endosperm wheat Fig13 8 % PAGE of PCR product amplified by Wx2B1 spe2 ( 3 A lane 16 18), bp cial tagged sequence Wx2B1, Briney [10 ] [3,4 ] A Note: 1) 11Marker ( 174) ; 21C1S1 ; 31Yangmai 158 ; 41N7A/ T7B ; 51DT4AS; 61N7D/ T7A ; 71N7D/ T7B ; 81Yangmai 158 ; 91Mianyang ; 101Neckawa ; 111Eradu ; 121Synthetic AABB/ DD ;131Mianyang ; 141Neixiang Baihuomai (b) ; 151Satanta ; Waxy wheat 1 ; Waxy wheat 2 ; Waxy endosperm wheat ; Mianyang ; 2 0 1Jiankang ; CIMMYT mv ; 221CIMMYTmv ; 231CIMMYTmv ; 241SD3523 2) Lane 9 13, null in Wx2B1 ; 14, 15 null in Wx2A1 and Wx2B1 ; null in all Wx protein. B Note : 1 ) 11Marker ( 174 ) ; 21SD3367 ; 31Mianyang ; 41Kanto107 ; 51Yangmai ; 61Yangmai ; 71CENT2 RO ; 81SAL259 ; 91Manital ; 101Russ ; 111Forge ; 121Ingot ; 131SD3650 ; 141SD3664 2) Lane 3, 5, 6, 9 null in Wx2B1 ; 4 null in Wx2A1 and Wx2B Wx2A1, Wx2D1 SSR Wx2A1, Wx2D1 SSR Shariflou [11 ] Wx cdna 3 SSR, 158 DNA 158 DT4AS bp ( 4 A lane 2, 3, 6), N7A/ T7B N7A/ T7D 265 bp ( 4 A lane 4, 5), 265 bp 7A Wx2A1 ( ) 265 bp, 230 bp ( 4 A lane 11, 13 17), 230 bp Wx2A1b, Wx2A1a Wx2A1b bp, SSR Wx2A1, N7D/ T7A N7D/ T7B, 204 bp ( 4 A lane 7, 8), 204 bp 7D Wx2D1 ( ) 7D 204 bp ( 4 A lane 10, 15 17), SDS2PAGE 4 B F 1, 158 Wx2A1 ( 4 B lane 3, 4) Wx2A1 Wx2B1 107 ( 4 B lane 7, 8) 3 ( 4 B lane 5, 6, 14, 15) F 1 ; Wx2B ( 4 B lane 9, 10) Wx2D1 (a) ( 4 B lane 11) F 1, Wx2A1, 6, bp ; Wx2B1 Eradu Wx2D1 (a) ( 4 B lane 12, 13), F 1 Wx2A1, 265 bp, 230 bp ; Wx2D1, 4 B 5, 11 15,, 204 bp SSR Wx2A1, Wx2D1 4 B 6 158/, 158, I 2 2KI F 1,,, Wx2A1 Wx2D1, Wx2A1

5 3 : ( T1 aestivum L1) Waxy 5 A B Note: 1 ) 1, 91Marker ( 174 ) ; 21C1S1 ; 31Yangmai 158 ; 41N7A/ T7B ; 51N7A/ T7D ; 61DT4AS; 71N7D/ T7A ; 81N7D/ T7B ; 101Jiangsu Baihuomai ; 111Kanto 107 ; 121IKE; 131Sturdy ; 141Sat2 anta ; 151Waxy wheat 1 ; 161Waxy wheat 2 ; 171Waxy endosperm wheat 2) Lane 10 null in Wx2D1 ; 11, 14 null in Wx2A1 and Wx2B1 ; 13 null in Wx2A1 ; null in all Wx protein 3 Note : 11Marker ; 21Yangmai 158 ; 3, 41Yangmai 158/ Holland wheat ; 51Yangmai 158/ Waxy wheat 2 ; 61Yangmai 158/ Waxy wheat 2 (false hybrid) ; 7, 81Yangmai 158/ Kanto 107 ; 9, 101Holland wheat/ Yangmai ; 111Holland wheat/ Neixiang Baihuomai ( a ) ; 12, 131Eradu/ Neixiang Baihuomai (a) ; 14,151Yangmai 158/ Waxy wheat 1 4 Wx2A1 Wx2D1 PCR 8 %PAGE Fig14 8 % PAGE of PCR product amplified by Wx2A1 and Wx2D1 [17 ] [6 900, 6 Wx2A1, 1 Wx2D1 ] 429, 7 Wx2A1, 29 Wx2B1, 3 Wx2D1, Wx2A1 Wx2B1 4 Yamamori [2 ], Wx2A1, 10 %, [2 Wx2B1, Wx2B1 48 % ] SDS2 PAGE 293, 14 ( ) Wx2B1, Wx2A1 Wx2D1,,,, SDS2PAGE Waxy 3, Wx,, Wx2B1 Wx2D1,,, 3 Wx,, PCR DNA,,, Wx2A1, Wx2A1a Wx2 A1b, Wx2A1, Wx2D1 SSR, Wx2A1 230 bp, Shariflou [11 ], [4 ] Shariflou [11 ] Wx cdna 3 SSR, 135, 7A Wx2A1 [4 ] SSR, Wx2A1 Wx2D1, Wx2A1 Wx2A1, Wx2D1 Wx2D1, Wx2A1 Wx2D1,, 2 % Wx2A1 STS Wx2A1 Wx2D1 SSR, Wx2A1, SSR Wx2D1 ; Wx2B1 STS Wx2B1,,,, Wx2B1, Wx2D1, Wx2B1 Wx2D1 : [1 ] Nakamura T, Yamamori M, Hirano H, et al. Identification of three waxy proteins in wheat ( Triticum aestivum L1) [J ]. Biochemical Genetics, 1993, 31 (1 2) : [2 ] Yamamori M, Nakamura T, Endo T R, et al. Waxy protein deficiency and chromosomal location of coding gene in common wheat [J ]. Theor Appl Genet, 1994, 89 : [3 ],,,. Wx [J ]., 2001, 9 (3) :

6 6 26 [4 ],,,. Wx [J ]., 2001, 28 (9) : [5 ] Yamamori M, Nakamura T, Kuroda A. Variations in the content of starch2granules bound protein among several Japanese cultivars of common wheat ( Triticum aestivum L1) [J ]. Euphitica, 1992, 64 : [6 ],,,. Waxy [J ]., 1999, 7 (1) : 1 9. [7 ] Demeke T, Hucl P, Nair R B, et al. Evaluation of Canadian and other wheats for waxy proteins [J ]. Cereal Chem, 1997, 74(4) : [8 ] Kiribuchi2Otobe C, Yasui T, Yanagisawa T, et al. Allelism test of hexaploid wheats from different sources [J ]. Breeding Science, 1998, 48 : [9 ] Nakamura T, Yamamori M, Hirano H, et al. Production of waxy (amylose2free) wheat [J ]. Mol Gen Genet, 1995, 248 : [ 10 ] Briney A, Wilson R, Potter H, et al. A PCR2based marker for selection of starch and potential noodle quality in wheat [J ]. Molecular Breeding, 1998, 4 : [11 ] Shariflou M R, Sharp P J. A polymorphic microsatellite in the 3 end of waxy genes of wheat, Triticum aestivum [J ]. Plant Breeding, 1999, 118 : [12 ] Shariflou M R, Hassani M E, Sharp P J. A PCR2based DNA marker for detection of mutant and normal alleles of the Wx2D1gene of wheat [J ]. Plant Breeding, 2001, 120 : [13 ],,,. Wx SDS2PAGE [J ]., 2000, 22 (3) : [14 ] Clark M S. [M].,. :, [15 ] Vrinten P, Nakamura T, Yamamori M. Molecular characterization of waxy mutations in wheat [J ]. Mol Gen Genet, 1999, 261 : [16 ] Murai J, Taira T, Othta D, et al. Isolation and characterization of the three waxy genes encoding the granule2bound starch synthase in hexaploid wheat [J ]. Gene, 1999, 234 : [17 ],,,. ( T1 aestivum) Waxy2D1 [J ]., 2000, 26 (3) : : 2004 ( ) 2004, 96 /, : 20 /, 240, : CN / S, : : 12 ( ) : , : : ; : ; : E2mail :