Molecular Breeding

, 25:145

Production of novel allelic variation for genes involved in starch biosynthesis through mutagenesis

  • F. Sestili
  • E. Botticella
  • Z. Bedo
  • A. Phillips
  • D. Lafiandra


Given the important role that starch plays in food and non-food uses of many crops, particularly wheat, efforts are being made to manipulate its composition through modification of the amylose/amylopectin ratio. Approaches used to achieve this goal include the manipulation of the genes involved in the starch biosynthetic pathway using natural or induced mutations and transgenic methods. The use of mutagenesis to produce novel allelic variation represents a powerful tool to increase genetic diversity and this approach seems particularly appropriate for starch synthase genes for which limited variation exists. In this work, an EMS-mutagenised population of bread wheat cv. Cadenza has been screened by combining SDS–PAGE analysis of granule bound starch proteins with a TILLING (Targeting Induced Local Lesions IN Genomes) approach at the gene level. In particular we have focused on two groups of synthase genes, those encoding the starch synthase II (Sgp-1) and those corresponding to the waxy proteins (Wx). SDS–PAGE analysis of granule bound proteins allowed the identification of single null genotypes associated with each of the three homoeologous loci. Molecular characterization of induced mutants has been performed using genome specific primer pairs for Sgp-1 and Wx genes. Additional novel allelic variation has also been detected at the different Sgp-1 homoeoloci by using a reverse genetic approach (TILLING). In particular single nucleotide substitutions, introducing a premature stop codon and creating amino acid substitutions, have been identified.


Wheat Mutagenesis TILLING Starch synthases 


  1. Ahloowalia BS, Maluszynski M, Nichterlein K (2004) Global impact of mutation-derived varieties. Euphytica 135:187–204CrossRefGoogle Scholar
  2. Bhattacharya M, Erazo-Castrejón SV, Doehlert DC, McMullen MS (2002) Staling of bread as affected by waxy wheat flour blends. Cereal Chem 79:178–182CrossRefGoogle Scholar
  3. Chao S, Sharp PJ, Worland AJ, Warham EJ, Koebner RMD, Gale MD (1989) RFLP-based genetic maps of wheat homoeologous group 7 chromosomes. Theor Appl Genet 78:495–504CrossRefGoogle Scholar
  4. Chibbar RN, Båga M, Ganeshan S, Hucl PJ, Limin A, Perron C, Ratnayaka I, Kapoor A, Verma V, Fowler DB (2005) Carbohydrate modification to add value to wheat grain. In: Chung OK, Lookhart GL (eds) Third international wheat quality conference, Manhattan, Kansas, USA, pp 75–84Google Scholar
  5. Comai L, Young K, Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, Henikoff S (2004) Efficient discovery of DNA polymorphisms in natural populations by Ecotilling. Plant J 37:778–786CrossRefPubMedGoogle Scholar
  6. Craig J, Lloyd JR, Tomlinson K, Barber L, Edwards A, Wang TL, Martin C, Hedley CL, Smith AM (1998) Mutations in the gene encoding starch synthase II profoundly alter amylopectin structure in pea embryos. Plant Cell 10:413–426CrossRefPubMedGoogle Scholar
  7. Ellis RP, Cochrane MP, Dale MFB, Duffus CM, Lynn A, Morrison IM, Prentice RDM, Swanston JS, Tiller SA (1998) Starch production and industrial use. J Sci Food Agric 77:289–311CrossRefGoogle Scholar
  8. Eriksson G (1970) The waxy character. Hereditas 63:180–204CrossRefGoogle Scholar
  9. Feiz L, Martin JM, Giroux MJ (2009) Creation and functional analysis of new Puroindoline alleles in Triticum aestivum. Theor Appl Genet 118:247–257CrossRefPubMedGoogle Scholar
  10. Greene EA, Codomo CA, Taylor NE, Henikoff JG, Till BJ, Reynolds SH, Enns LC, Burtner C, Johnson JE, Odden AR, Comai L, Henikoff S (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164:731–740PubMedGoogle Scholar
  11. Maluszynski M, Szarejko I, Maluszynska J (2001) Induced mutations in wheat. In: Bonjean AP, Angus WJ (eds) The world wheat book. A history of wheat breeding, pp 939–977Google Scholar
  12. McCallum CM, Comai L, Greene EA, Henikoff S (2000) Targeted screening for induced mutations. Nat Biotechnol 18:455–457CrossRefPubMedGoogle Scholar
  13. Mohammadkhani A, Stoddard FL, Marshall DR, Uddin MN, Zhao X (1999) Starch extraction and amylose analysis from half seeds. Starch/Staerke 51:62–66CrossRefGoogle Scholar
  14. Morell MK, Kosar-Hashemi B, Cmiel M, Samuel MS, Chandler P, Rahman S, Buleon A, Batey IL, Li ZY (2003) Barley sex6 mutants lack starch synthase IIa activity and contain a starch with novel properties. Plant J 34:172–184CrossRefGoogle Scholar
  15. Murai J, Taira T, Ohta D (1999) Isolation and characterisation of the three Waxy genes encoding the granule-bound starch synthase in hexaploid wheat. Gene 234:71–79CrossRefPubMedGoogle Scholar
  16. Nakamura T, Yamamori M, Hirano H, Hidaka S, Nagamine T (1995) Production of waxy (amylose-free) wheats. Mol Gen Genet 248:253–259CrossRefPubMedGoogle Scholar
  17. Parry MAJ, Madgwick PJ, Bayon C, Tearall K, Hernandez-Lopez A, Baudo M, Rakszegi M, Hamada W, Al-Yassin A, Ouabbou H, Labhilili M, Phillips AL (2009) Mutation discovery for crop improvement. J Exp Bot (in press)Google Scholar
  18. Rahman S, Li Z, Regina A, Kosar-Hashemi B, McMaugh S, Konik-Rose C, Morell M (2005) Genetic control of wheat starch biosynthesis. Wheat Inf Serv 100:77–87Google Scholar
  19. Rahman S, Bird A, Regina A, Li Z, Ral JP, McMaugh S, Topping D, Morell M (2007) Resistant starch in cereals: exploiting genetic engineering and genetic variation. J Cereal Sci 46:251–260CrossRefGoogle Scholar
  20. Sears ER, Sears LMS (1978) The telocentric chromosomes of common wheat. In: Ramanujan S (ed) 5th International Wheat Genetics Symposium, New Delhi, pp 389–407Google Scholar
  21. Shimbata T, Nakamura T, Vrinten P, Saito M, Yonemaru J, Seto Y, Yasuda H (2005) Mutations in wheat starch synthase II genes and PCR-based selection of a SGP-1 null line. Theor Appl Genet 111:1072–1079CrossRefPubMedGoogle Scholar
  22. Slade AJ, Knauf VC (2005) TILLING moves beyond functional genomics into crop improvement. Transgenic Res 14:109–115CrossRefPubMedGoogle Scholar
  23. Slade AJ, Fuerstenberg SI, Loeffler D, Steine MN, Facciotti D (2005) A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat Biotechnol 23:75–81CrossRefPubMedGoogle Scholar
  24. Soh HN, Sisson MJ, Turner M (2006) Effect of starch granule size distribution and elevated amylose content on durum dough rheology and spaghetti cooking quality. Cereal Chem 83:513–519CrossRefGoogle Scholar
  25. Tai T, Tanksley S (1991) A rapid and inexpensive method for isolation of total DNA from dehydrated plant tissue. Plant Mol Biol Rep 8:297–303CrossRefGoogle Scholar
  26. Till BJ, Zerr T, Comai L, Henikoff S (2006) A protocol for TILLING and ecotilling in plants and animals. Nat Prot 1:2465–2477CrossRefGoogle Scholar
  27. Topping D (2007) Cereal complex carbohydrates and their contribution to human health. J Cereal Sci 46:220–229CrossRefGoogle Scholar
  28. Urbano M, Margiotta B, Colaprico G, Lafiandra D (2002) Waxy protein in diploid, tetraploid and hexaploid wheats. Plant Breed 121:1–5CrossRefGoogle Scholar
  29. Van Deynze A, Stoffel K (2006) High-throughput DNA extraction from seeds. Seed Sci Technol 34:741–745Google Scholar
  30. Van Hung P, Yasui T, Maeda T, Morita N (2007) Dough properties and breadmaking qualities of whole waxy wheat flour and effects of additional enzymes. J Sci Food Agric 87:2538–2543CrossRefGoogle Scholar
  31. Yamamori M, Endo TR (1996) Variation of starch granule proteins and chromosome mapping of their coding genes in common wheat. Theor Appl Genet 93:275–281CrossRefGoogle Scholar
  32. Yamamori M, Nakamura T, Endo TR, Nagamine T (1994) Waxy protein deficiency and chromosomal location of coding genes in common wheat. Theor Appl Genet 89:179–184Google Scholar
  33. Yamamori M, Fujita S, Hayakawa K, Matsuki J, Yasui T (2000) Genetic elimination of a starch granule protein, SGP-1, of wheat generates an altered starch with apparent high amylose. Theor Appl Genet 101:21–29CrossRefGoogle Scholar
  34. Yoo SH, Jane JL (2002) Structural and physical characteristics of waxy and other wheat starches. Carbohydr Polym 49:297–305CrossRefGoogle Scholar
  35. Zhang X, Colleoni C, Ratushna V, Sirghie-Colleoni M, James MG, Myers AM (2004) Molecular characterization demonstrates that the Zea mays gene sugary2 codes for the starch synthase isoform SSIIa. Plant Mol Biol 54:865–879CrossRefPubMedGoogle Scholar
  36. Zhao XC, Sharp PJ (1996) An improved 1-D SDS–PAGE method for the identification of three bread wheat waxy proteins. J Cereal Sci 23:191–193CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • F. Sestili
    • 1
  • E. Botticella
    • 1
  • Z. Bedo
    • 2
  • A. Phillips
    • 3
  • D. Lafiandra
    • 1
  1. 1.Department of Agrobiology and AgrochemistryUniversity of TusciaViterboItaly
  2. 2.Agricultural Research Institute of the Hungarian Academy of SciencesMartonvasarHungary
  3. 3.Rothamsted ResearchHarpendenUK

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