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Identification of allelic variations of puroindoline genes controlling grain hardness in wheat using a modified denaturing PAGE

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Abstract

Puroindoline genes (Pina and Pinb) form the molecular basis of wheat grain hardness or texture. Variations in either gene are reported to be associated with grain hardness in wheat. Here, a modified denaturing PAGE was used to detect Pina and Pinb allelic variations in 102 common wheat cultivars and other species related to wheat. Two variations of Pina (Pina-D1b and Pina-D1p) and five variations of Pinb (null, Pinb-D1b, Pinb-D1u, Pinb-D1v and Pinb-D1w) were identified in common (T. aestivum) and spelt wheats (T. aestivum ssp. spelta). No allelic variation was found in Tibet semi-wild wheat (T. aestivum ssp. tibetanum), club wheat (T. compactum), or Aegilops  tauschii. Puroindoline genes were absent in wild␣emmer (T. turgidum var. dicoccoides). The sequencing results of the PCR fragments from Pina and Pinb (except for null type) indicated allelic variants carrying single base mutation, such as Pina-D1p, Pinb-D1u, Pinb-D1v and Pinb-D1w were novel types. Our results showed that the modified PAGE used in this study provided a satisfactory resolving power for identifying single nucleotide mutations; therefore, it is a practical and simple tool to study allelic variation of Pina and Pinb in wheat and related species.

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Abbreviations

ARMS:

Amplification Refractory Mutation System

ASO:

Allele Specific Oligonuceotide

ASP:

Allelic Specific Primers

DGGE:

Denaturing Gradient Gel Electrophoresis

SSCP:

Single Strand Conformation Polymorphism

STS:

Sequenced Tagged Site

SDS-PAGE:

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis

A-PAGE:

Acidic Polyacrylamide Gel Electrophoresis

2-D PAGE:

Two-dimensional Polyacrylamide Gel Electrophoresis

TBE:

Tris-Boric acid-EDTA

Reference

  • American Association of Cereal Chemists (1995). Approved methods of the AACC, 9th Ed. Wheat hardness as determined by near-infrared reflectance, Method 39-70A. The AACC, St. Paul, Minnesota

    Google Scholar 

  • Amoroso MG, Longobardo L, Capparelli R (2004) Real time RT-PCR and flow cytometry to investigate wheat kernel hardness: role of puroindolines genes and proteins. Biotech Lett 26:1731–1737

    Article  CAS  Google Scholar 

  • Bettge AD, Morris CF (2000) Relationship among grain hardness, pentosan fraction and end-use quality of wheat. Cereal Chem 77:241–247

    CAS  Google Scholar 

  • Blochet JE, Chevalier C, Forest E, Pebay-Peyroula E, Gautier MF, Joudrier P, Pézolet M, Marion D (1993) Complete amino acid sequence of puroindoline, a new basic and cystine-rich protein with a unique tryptophan-rich domain, isolated from wheat endosperm by tritonx-114 phase portioning. FEBS Lett 329:336–340

    Article  PubMed  CAS  Google Scholar 

  • Capparelli R, Borriello G, Giroux MJ, Amoroso MG (2003) Puroindoline A-gene expression is involved in association of puroindolines to starch. Theor Appl Genet 107:1463–1468

    Article  PubMed  CAS  Google Scholar 

  • Corona V, Gazza L, Boggini G, Pogna NE (2001) Variation in friabilin composition as determined by A-PAGE fraction and PCR amplification, and its relationship to grain hardness in bread wheat. J Cereal Sci 34:243–250

    Article  CAS  Google Scholar 

  • Chen F, He ZH, Xia XC, Xia LQ, Zhang XY, Lillemo M, Morris CF (2006) Molecular and biochemical characterization of puroindoline a and b alleles in Chinese landraces and historical cultivars. Theor Appl Genet 112:400–409

    Article  PubMed  CAS  Google Scholar 

  • Du CF, Liu HM, Li RZ, Li PB (2003) Application of single nucleotide polymorphism in crop genetics and improvement. Hereditas (Beijing) 25:735–739

    CAS  Google Scholar 

  • Dubreil L, Compoint JP, Marion D (1997) Interaction of puroindolines with wheat flour polar lipids determining their foaming properties. J Agric Food Chem 45:108–116

    Article  CAS  Google Scholar 

  • Dubcovsky J, Tranquilli G, Lijavetzky D, Muzzi G (1999) Genetic and physical characterization of grain texture -related loci in diploid wheat. Mol Gen Genet 262:846–850

    Article  PubMed  Google Scholar 

  • Galande AA, Tiwari R, Ammiraju JSS, Santra DK, Lagu MD, Rao VS, Gupta VS, Misra BK, Nagarajan S, Ranjekar PK (2001) Genetic analysis of kernel hardness in bread wheat using PCR-based markers. Theor Appl Genet 103:601–606

    Article  CAS  Google Scholar 

  • Gautier MF, Aleman ME, Guirao A, Marion D, Joudrier P (1994) Triticum  aestivum puroindolines, two basic cystine-rich seed proteins: cDNA sequence and developmental gene expression. Plant Mol Biol 25:43–57

    Article  PubMed  CAS  Google Scholar 

  • Gautier MF, Cosson P, Guirao A, Alary R, Joudrier P (2000) Puroindoline genes are highly conserved in diploid ancestor wheats and related species but absent in tetraploid Triticum species. Plant Sci 153:81–91

    Article  CAS  Google Scholar 

  • Gazza L, Nocente F, Ng PKW, Pogna NE (2005) Genetic and biochemical analysis of common wheat cultivars lacking puroindoline a. Theor Appl Genet 110:470–478

    Article  PubMed  CAS  Google Scholar 

  • Giroux MJ, Morris CF (1997) A glycine to serine change in puroindoline b is associated with wheat grain hardness and low levels of starch surface fribilin. Theor Appl Genet 95:857–864

    Article  CAS  Google Scholar 

  • Giroux MJ, Morris CF (1998) Wheat grain hardness results from highly conserved mutations in the friabilin component puroindoline a and b. Proc Natl Acad Sci USA 95:6262–6266

    Article  PubMed  CAS  Google Scholar 

  • Greenwell P, Schofield JD (1986) A starch granule protein associated with endosperm softness in wheat. Cereal Chem 63:379–380

    CAS  Google Scholar 

  • Greenblatt GA, Bettge AD, Morris CF (1995) The relationship among endosperm texture, friabilin occurrence and bound polar lipids on wheat starch. Cereal Chem 72:172–176

    CAS  Google Scholar 

  • Guidet F, Rogowsky R, Taylor C, Song W, Langridge P (1991) Cloning and characterizations of a new rye-specific repeat sequence. Genome 34:81–87

    Google Scholar 

  • Guo SH, He ZH, Xia LQ, Wang HG, Zhang QZ (2004) Detection of allelic variation for grain hardness in Chinese spring wheat by STS marker. Sci Agri Sin 37:1797–1803

    CAS  Google Scholar 

  • Hong BH, Rubenthaler GL, Allan RE (1989) Wheat pentosans I cultivar variation and relationship to kernel hardness. Cereal Chem 66:369–373

    CAS  Google Scholar 

  • Igrejas G, Leroy P, Charmet G, Gaborit T, Marion D, Branlard G (2002) Mapping QTLs for grain hardness and puroindoline content in wheat (Triticum  aestivum L.). Theor Appl Genet 106:19–27

    PubMed  CAS  Google Scholar 

  • Law CN, Young CF, Brown JWS, Snape JW, Worland JW (1978) The study of grain protein control in wheat using whole chromosome substitution lines. In: Seeds protein improvement by nuclear techniques. International Atomic Energy Agency, Vienna, Austria, pp 483–502

  • Lillemo M, Morris CF (2000) A leucine to proline mutation in puroindoline b is frequently present in hard wheat from Northern Europe. Theor Appl Genet 100:1100–1107

    Article  CAS  Google Scholar 

  • Marion D, Gautier MF, Joudrier P, Ptak M, Pezolet M, Forest E, Clark DC, Broekaert W (1994) Structure and function of wheat lipid binding proteins. In: Martino al Cimino S (ed) Wheat kernel proteins – molecular and functional aspects. Univ. Degli Studi Della Tuscia, Viterbo, Italy, pp 175–180

    Google Scholar 

  • McIntosh RA, Devos KM, Dubcovsky J, Rogers WJ, Morris CF, Appels R, Anderson OD (2005) Catalogue of gene symbols for wheat. http://wheat.pw.usda.gov/ggpages/wgc/2005 upd.html

  • Morris CF, Greenblatt GA, Bettge AD, Malkawi HI (1994) Isolation and characterization of multiple forms of friabilin. J Cereal Sci 20:167–174

    Article  CAS  Google Scholar 

  • Morris CF, Rose SP (1996) Wheat. In: Henry RJ, Kettlewell PS (eds) Cereal grain quality. Chapman Hall, London, pp 3–54

    Google Scholar 

  • Morris CF (2002) Puroindolines: the molecular genetic basis of wheat grain hardness. Plant Mol Bio 48:633–647

    Article  CAS  Google Scholar 

  • Myers RM, Maniatis T, Lerman L (1987) Detection and localization of single base changes by denaturing gradient gel electrophoresis. Meth Enzym 155:501–527

    Article  PubMed  CAS  Google Scholar 

  • Myers RM, Shielfield VC, Cox DR (1990) Mutation detection by PCR, GC-Clamps and denaturing gradient gel electrophoresis. In: Erich HA (ed) PCR technology: principles and applications for DNA amplification. Stockton Press, New York, pp 71–88

    Google Scholar 

  • Oda S (1994) Two-dimensional electrophoretic analysis of friabilin. Cereal Chem 71:394–395

    CAS  Google Scholar 

  • Pomeranz Y, Williams PC (1990) Wheat hardness: its genetic, structural, and biochemical background, measurement, and significance. In: Pomeranz Y (ed) Advances in cereal science and technology, vol X. AACC, St. Paul, MN, USA, pp 471–548

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 327–329, 886–887

  • Sourdille P, Perretant MR, Charmet G, Leroy P, Gautier MF, Joudrier P, Nelson JC, Sorrells ME, Bernard M (1996) Linkage between RFLP markers and genes affecting kernel hardness in wheat. Theor Appl Genet 93:580–586

    CAS  Google Scholar 

  • Tippless KH, Kilborn RH, Preston KR (1994) Bread-wheat quality defined. In: Bushuk W, Rasper VF (eds) Wheat: production, properties and quality. Blackwell Academic and Professional, Chapman & Hall, Glasgow, pp 25–35

    Google Scholar 

  • Turnbull KM, Rahman S (2002) Endosperm texture in wheat. J Cereal Sci 36:327–337

    Article  CAS  Google Scholar 

  • Turner AS, Bradburne RP, Fish L, Snape JW (2004) New quantitative trait loci influencing grain texture and protein content in bread wheat. J Cereal Sci 40:51–60

    Article  CAS  Google Scholar 

  • Zhang XN, Zhang YF (2001) Determination low molecular weight polypeptide by Glycine-SDS-PAGE. Biotechnology 11:10–12

    Google Scholar 

  • Zhou YH, He ZH, Yan J, Zhang Y, Wang DS, Zhou GY (2002) Distribution of grain hardness in Chinese wheat and genetic analysis. Sci Agri Sin 35:1177–1185

    CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. Shi-he Xiao (Institute of Crop Sciences in CAAS) and Prof. Qi-xin Sun (CAU) for kindly providing materials. We also thank the two anonymous reviewers for valuable comments on this manuscript and Dr. Hong-Wei Cai (Forage Crop Research Institute, Japan Grassland Agriculture & Forage Seed Association) for helpful discussion on the manuscript. This work was supported by a grant from National Natural Foundation of China (Number 30270823, 30471076).

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Authors and Affiliations

  1. Beijing Key Laboratory of Crop Genetic Improvement, Key Laboratory of Crop Genomics & Genetic Improvement, Ministry of Agriculture, the People’s Republic of China, College of Agronomy and Biotechnology, China Agricultural University (CAU), 2 Yuanmingyuan West Road, Beijing, 100094, P.R. China

    Cheng Chang, Jie Xu, Weihua Li, Guangtian Liu, Mingshan You & Baoyun Li

  2. Institute of Crop Sciences/National Wheat Improvement Center, Chinese Academy of Agricultural Sciences (CAAS), 12 Zhongguancun South Street, Beijing, 100081, P.R. China

    Cheng Chang & Haiping Zhang

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  1. Cheng Chang
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  2. Haiping Zhang
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  3. Jie Xu
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Correspondence to Baoyun Li.

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Chang, C., Zhang, H., Xu, J. et al. Identification of allelic variations of puroindoline genes controlling grain hardness in wheat using a modified denaturing PAGE. Euphytica 152, 225–234 (2006). https://doi.org/10.1007/s10681-006-9204-6

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  • DOI: https://doi.org/10.1007/s10681-006-9204-6

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