, Volume 141, Issue 4–6, pp 227–238 | Cite as

Characterization and expression analysis of waxy alleles in barley accessions

  • Jian Ma
  • Qian-Tao Jiang
  • Quan-Zhi Zhao
  • Shan Zhao
  • Xiu-Jin Lan
  • Shou-Fen Dai
  • Zhen-Xiang Lu
  • Chunji Liu
  • Yu-Ming Wei
  • You-Liang Zheng


Granule Bound Starch Synthase I (GBSS I) encoded by the waxy gene plays an important role in accumulating amylose during the development of starch granules in barley. In this study, we isolated and characterized waxy alleles of three waxy (GSHO 908, GSHO 1828 and NA 40) and two non-waxy barley accessions (PI 483237 and CIho 15773), estimated the expression patterns of waxy genes via Real-time quantitative PCR (RT-qPCR), investigated promoter activity by analyzing promoter-GUS expression, and examined possible effects of waxy alleles on starch granule morphology in barley accessions by scanning electron microscopy (SEM). A 193-bp insertion in intron 1, a 15-bp insertion in the coding region, and some single nucleotide polymorphic sites were detected in the waxy barley accessions. In addition, a 397-bp deletion containing the TATA box, transcription starting point, exon 1 and partial intron 1 were also identified in the waxy barley accessions. RT-qPCR analysis showed that waxy accessions had lower waxy expression levels than those of non-waxy accessions. Transient expression assays showed that GUS activity driven by the 1,029-bp promoter of the non-waxy accessions was stronger than that driven by the 822-bp promoter of the waxy accessions. SEM revealed no apparent differences of starch granule morphology between waxy and non-waxy accessions. Our results showed that the 397-bp deletion identified in the waxy barley accessions is likely responsible for the reduction of waxy transcript, leading to lower concentrations of GBSS I protein thus lower amylose content.


Barley GBSS I Waxy Amylose Real-time quantitative PCR 



This work was supported by the National Natural Science Foundation of China (31230053, 31000167 and 31171556) and China Transgenic Research Program (2011ZX08002-001,004 and 005) the National Basic Research Program of China (973 Program 2010CB134400).

Supplementary material

10709_2013_9721_MOESM1_ESM.tif (1 mb)
Supplementary material 1 (TIFF 1062 kb)
10709_2013_9721_MOESM2_ESM.tif (135 kb)
Supplementary material 2 (TIFF 135 kb)
10709_2013_9721_MOESM3_ESM.tif (407 kb)
Supplementary material 3 (TIFF 406 kb)
10709_2013_9721_MOESM4_ESM.tif (118 kb)
Supplementary material 4 (TIFF 118 kb)
10709_2013_9721_MOESM5_ESM.doc (22 kb)
Supplementary material 5 (DOC 22 kb)
10709_2013_9721_MOESM6_ESM.doc (28 kb)
Supplementary material 6 (DOC 28 kb)
10709_2013_9721_MOESM7_ESM.doc (77 kb)
Supplementary material 7 (DOC 77 kb)


  1. Bhatty R (1993) Nonmalting uses of barley, Barley: Chemistry and Technology. In: MacGregor AW and Bhatty RS (eds), Am. Assoc. Cereal Chem, St. Paul, MN, pp 355–417Google Scholar
  2. Bird AR, Brown IL, Topping DL (2000) Starches, resistant starches, the gut microflora and human health. Curr Issues Intestinal Microbiol 1:25–37Google Scholar
  3. Boscari A, Clement M, Volkov V, Golldack D, Hybiak J, Miller AJ, Amtmann A, Fricke W (2009) Potassium channels in barley: cloning, functional characterization and expression analyses in relation to leaf growth and development. Plant, Cell Environ 32:1761–1777CrossRefGoogle Scholar
  4. Cai XL, Wang ZY, Xing YY, Zhang JL, Hong MM (1998) Aberrant splicing of intron 1 leads to the heterogeneous 5′ UTR and decreased expression of waxy gene in rice cultivars of intermediate amylose content. Plant J 14:459–465PubMedCrossRefGoogle Scholar
  5. Chen PY, Wang CK, Soong SC, To KY (2003) Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants. Mol Breed 11:287–293CrossRefGoogle Scholar
  6. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31:3497–3500PubMedCrossRefGoogle Scholar
  7. Denyer K, Johnson P, Zeeman S, Smith AM (2001) The control of amylose synthesis. J Plant Physiol 158:479–487CrossRefGoogle Scholar
  8. Domon E, Fuijita M, Ishikawa N (2002a) The insertion/deletion polymorphisms in the waxy gene of barley genetic resources from East Asia. Theor Appl Genet 104:132–138PubMedCrossRefGoogle Scholar
  9. Domon E, Saito A, Takeda K (2002b) Comparison of the waxy locus sequence from a non-waxy strain and two waxy mutants of spontaneous and artificial origins in barley. Genes Genet Syst 77:351–359PubMedCrossRefGoogle Scholar
  10. Echt CS, Schwartz D (1981) Evidence for the inclusion of controlling elements within the structural gene at the waxy locus in maize. Genetics 99:275–284PubMedGoogle Scholar
  11. Ezcurra I, Ellerström M, Wycliffe P, Stålberg K, Rask L (1999) Interaction between composite elements in the napA promoter: both the B-box ABA-responsive complex and the RY/G complex are necessary for seed-specific expression. Plant Mol Biol 40:699–709PubMedCrossRefGoogle Scholar
  12. Fukunaga K, Kawase M, Kato K (2002) Structural variation in the Waxy gene and differentiation in foxtail millet [Setaria italica (L.) P. Beauv.]: implications for multiple origins of the waxy phenotype. Mol Genet Genomics 268:214–222PubMedCrossRefGoogle Scholar
  13. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27:297–300PubMedCrossRefGoogle Scholar
  14. Hunt HV, Denyer K, Packman LC, Jones MK, Howe CJ (2010) Molecular basis of the waxy endosperm starch phenotype in broomcorn millet (Panicum miliaceum). Mol Biol Evol 27:1478–1494PubMedCrossRefGoogle Scholar
  15. James MG, Denyer K, Myers AM (2003) Starch synthesis in the cereal endosperm. Curr Opin Plant Biol 6:215–222PubMedCrossRefGoogle Scholar
  16. Jeng TL, Wang CS, Tseng TH, Wu MT, Sung JM (2009) Nucleotide polymorphisms in the waxy gene of NaN3–induced waxy rice mutants. J Cereal Sci 49:112–116CrossRefGoogle Scholar
  17. Jiang QT, Liu T, Ma J, Wei YM, Lu ZX, Lan XJ, Dai SF, Zheng YL (2012a) Characterization of barley Prp1 gene and its expression during seed development and under abiotic stress. Genetica 139:1283–1292Google Scholar
  18. Jiang QT, Ma J, Zhao S, Zhao QZ, Lan XJ, Dai SF, Lu ZX, Zheng YL, Wei YM (2012b) Characterization of HMW-GSs and their gene inaction in tetraploid wheat. Genetica 140:325–335Google Scholar
  19. Jobling S (2004) Improving starch for food and industrial applications. Curr Opin Plant Biol 7:210–218PubMedCrossRefGoogle Scholar
  20. Kluth A, Sprunck S, Becker D, Lörz H, Lütticke S (2002) 5′ deletion of a gbss1 promoter region from wheat leads to changes in tissue and developmental specificities. Plant Mol Biol 49:665–678CrossRefGoogle Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  22. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30:325–327PubMedCrossRefGoogle Scholar
  23. Li J, Vasanthan T, Rossnagel B, Hoover R (2001) Starch from hull-less barley: i. Granule morphology, composition and amylopectin structure. Food Chem 74:395–405CrossRefGoogle Scholar
  24. Liu S, Bao Y (2009) Effects of copy number of an octopine synthase enhancer element and its distance from the TATA box on heterologous expression in transgenic tobacco. Acta Physiologiae Plantarum 31:705–710CrossRefGoogle Scholar
  25. Liu WX, Liu HL, Chai ZJ, Xu XP, Song YR, Qu LQ (2010) Evaluation of seed storage-protein gene 5′ untranslated regions in enhancing gene expression in transgenic rice seed. Theor Appl Genet 121:1267–1274PubMedCrossRefGoogle Scholar
  26. Ma J, Jiang QT, Wei YM, Andre L, Lu ZX, Chen GY, Liu YX, Zheng YL (2010) Molecular characterization and comparative analysis of two waxy alleles in barley. Genes Genom 32:513–520CrossRefGoogle Scholar
  27. Murray M, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4326PubMedCrossRefGoogle Scholar
  28. Patron NJ, Smith AM, Fahy BF, Hylton CM, Naldrett MJ, Rossnagel BG, Denyer K (2002) The altered pattern of amylose accumulation in the endosperm of low-amylose barley cultivars is attributable to a single mutant allele of granule-bound starch synthase I with a deletion in the 5′-non-coding region. Plant Physiol 130:190–198PubMedCrossRefGoogle Scholar
  29. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 29:e45–e45PubMedCrossRefGoogle Scholar
  30. Radchuk VV, Borisjuk L, Sreenivasulu N, Merx K, Mock HP, Rolletschek H, Wobus U, Weschke W (2009) Spatiotemporal profiling of starch biosynthesis and degradation in the developing barley grain. Plant Physiol 150:190–204PubMedCrossRefGoogle Scholar
  31. Regina A, Blazek J, Gilbert E, Flanagan BM, Gidley MJ, Cavanagh C, Ral J-P, Larroque O, Bird AR, Li Z, Morell MK (2012) Differential effects of genetically distinct mechanisms of elevating amylose on barley starch characteristics. Carbohydr Polym 89:979–991CrossRefGoogle Scholar
  32. Rogers H, Bate N, Combe J, Sullivan J, Sweetman J, Swan C, Lonsdale D, Twell D (2001) Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10. Plant Mol Biol 45:577–585PubMedCrossRefGoogle Scholar
  33. Rohde W, Becker D, Salamini F (1988) Structural analysis of the waxy locus from Hordeum vulgare. Nucleic Acids Res 16:7185PubMedCrossRefGoogle Scholar
  34. Rösti S, Rudi H, Rudi K, Opsahl-Sorteberg HG, Fahy B, Denyer K (2006) The gene encoding the cytosolic small subunit of ADP-glucose pyrophosphorylase in barley endosperm also encodes the major plastidial small subunit in the leaves. J Exp Bot 57:3619–3626PubMedCrossRefGoogle Scholar
  35. Rushton PJ, Reinstädler A, Lipka V, Lippok B, Somssich IE (2002) Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen-and wound-induced signaling. Plant Cell 14:749–762PubMedCrossRefGoogle Scholar
  36. Sattler SE, Singh J, Haas EJ, Guo L, Sarath G, Pedersen JF (2009) Two distinct waxy alleles impact the granule-bound starch synthase in sorghum. Mol Breeding 24:349–359CrossRefGoogle Scholar
  37. Soong R, Ruschoff J, Tabiti K (2000) Detection of colorectal micrometastasis by quantitative RT-PCR of cytokeratin 20 mRNA. Roche Diagnostics internal publicationGoogle Scholar
  38. Swanston J, Ellis R, Stark J (1995) Effects on grain and malting quality of genes altering barley starch composition. J Cereal Sci 22:265–273CrossRefGoogle Scholar
  39. Swapna L, Khurana R, Vijaya Kumar S, Tyagi A, Rao K (2011) Pollen-specific expression of Oryza sativa indica pollen allergen gene (OSIPA) promoter in rice and Arabidopsis transgenic systems. Mol Biotechnol 48:49–59PubMedCrossRefGoogle Scholar
  40. Tang H, Watanabe K, Mitsunaga T (2002) Structure and functionality of large, medium and small granule starches in normal and waxy barley endosperms. Carbohydr Polym 49:217–224CrossRefGoogle Scholar
  41. Tester RF, Karkalas J, Qi X (2004) Starch-composition, fine structure and architecture. J Cereal Sci 39:151–165CrossRefGoogle Scholar
  42. Van Hung P, Maeda T, Morita N (2006) Waxy and high-amylose wheat starches and flours—characteristics, functionality and application. Trends Food Sci Technol 17:448–456CrossRefGoogle Scholar
  43. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:research0034Google Scholar
  44. Weschke W, Panitz R, Gubatz S, Wang Q, Radchuk R, Weber H, Wobus U (2003) The role of invertases and hexose transporters in controlling sugar ratios in maternal and filial tissues of barley caryopses during early development. Plant J 33:395–411PubMedCrossRefGoogle Scholar
  45. Yanagisawa T, Kiribuchi-Otobe C, Yoshida H (2001) An alanine to threonine change in the Wx-D1 protein reduces GBSS I activity in waxy mutant wheat. Euphytica 121:209–214CrossRefGoogle Scholar
  46. Zeeman SC, Kossmann J, Smith AM (2010) Starch: its metabolism, evolution, and biotechnological modification in plants. Annu Rev Plant Biol 61:209–234PubMedCrossRefGoogle Scholar
  47. Zhu CM (2009) Research on genetic diversity of waxy germplasm resources and wx gene in Chinese barley. Chinese Academy of Agricultural Sciences Publishing, Beijing (in Chinese)Google Scholar
  48. Zhu T, Jackson DS, Wehling RL, Geera B (2008) Comparison of amylose determination methods and the development of a dual wavelength Iodine binding technique 1. Cereal Chem 85:51–58CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Jian Ma
    • 1
  • Qian-Tao Jiang
    • 1
  • Quan-Zhi Zhao
    • 1
  • Shan Zhao
    • 1
  • Xiu-Jin Lan
    • 1
  • Shou-Fen Dai
    • 1
  • Zhen-Xiang Lu
    • 2
  • Chunji Liu
    • 3
  • Yu-Ming Wei
    • 1
  • You-Liang Zheng
    • 4
  1. 1.Triticeae Research InstituteSichuan Agricultural UniversityChengduChina
  2. 2.Lethbridge Research CentreAgriculture and Agri-Food CanadaLethbridgeCanada
  3. 3.CSIRO Plant IndustrySt. LuciaAustralia
  4. 4.Key Laboratory of Southwestern Crop Germplasm Utilization, Ministry of AgricultureSichuan Agricultural UniversityYa’anChina

Personalised recommendations