Molecular Breeding

, Volume 25, Issue 4, pp 623–635 | Cite as

Waxy strains of three amaranth grains raised by different mutations in the coding region

  • Young-Jun Park
  • Kazuhiro Nemoto
  • Tomotaro Nishikawa
  • Kenichi Matsushima
  • Mineo Minami
  • Makoto Kawase


Waxy strains raised by waxy mutation have been found for three amaranth grains. Three genes encoding waxy protein were isolated from Amaranthus caudatus (Wx-ca), A. cruentus (Wx-cr), and A. hypochondriacus (Wx-hy). Sequence analysis indicated that the Wx-ca, Wx-cr, and Wx-hy genes contained the same exon (13 exons) and intron (12 introns) structure. The lengths of the Wx-ca, Wx-cr, and Wx-hy genes were 3,236, 3,237, and 3,225 bp, respectively. The alignment of the coding sequence of the three Waxy genes showed 12 polymorphic sites, including 11 single nucleotide polymorphisms (SNPs) (in exons 10 and 12 and introns 1, 3, 4, 9, and 11) and 5 deletions or insertions (indels) (in introns 4, 9, and 11). In particular, major polymorphism was detected in 8- and 3-bp indels in intron 4. Moreover, the mutation in the waxy alleles (wx-ca, wx-cr, and wx-hy) of all three species was also isolated and characterized. Comparison of coding sequences of the three Waxy genes and their waxy alleles indicated one base insertion (wx-ca: insert of T base in exon 8) and a base substitution (wx-cr: a G-to-T base substitution in exon 10; wx-hy: a G-to-A base substitution in exon 6), which occurred as internal termination codon in the three Waxy genes, suggesting the involvement of a nonsense or frameshift mutation. Therefore, these different mutations in coding regions were considered to be the cause of the waxy (amylose-free) phenotype.


Grain amaranth Waxy protein Waxy mutant Nonsense mutation 



We express our sincere thanks to Dr. D. Brenner of USDA-ARS-MWA-PIRU; Iowa State University, USA, for providing the accessions used in this experiment.


  1. Ainsworth C, Gale M, Baird S (1983) The genetics of β-amylase isozymes in wheat. Theor Appl Genet 66:39–49CrossRefGoogle Scholar
  2. Arciga-Reyes L, Wootton L, Kieffer M, Davies B (2006) UPF1 is required for nonsense-mediated mRNA decay (NMD) and RNAi in arabidopsis. Plant J 47:480–489CrossRefPubMedGoogle Scholar
  3. Breathnach R, Chambon P (1981) Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem 50:349–383CrossRefPubMedGoogle Scholar
  4. Breene W (1991) Food uses of grain amaranth. Cereal Foods World 36:426–430Google Scholar
  5. Bureau T, Wessler S (1992) Tourist: a large family of small inverted repeat elements frequently associated with maize genes. Plant Cell 4:1283–1294CrossRefPubMedGoogle Scholar
  6. Cai X, Wang Z, Xing Y, Zhang J, Hong M (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 14:459–465CrossRefPubMedGoogle Scholar
  7. Camirand A, St-Pierre B, Marineau C, Brisson N (1990) Occurrence of a copia-like transposable element in one of the introns of the potato starch phosphorylase gene. Mol Gen Genet 224:33–39CrossRefPubMedGoogle Scholar
  8. Campbell WH, Gowri G (1990) Codon usage in higher plants, green algae, and cyanobacteria. Plant Physiol 92:1–11CrossRefPubMedGoogle Scholar
  9. Chan KF, Sun M (1997) Genetic diversity detected by isozyme and RAPD analysis of crop and wild species of amaranthus. Theor Appl Genet 95:865–873CrossRefGoogle Scholar
  10. Clark J, Robertson M, Ainsworth C (1991) Nucleotide sequence of a wheat (Triticum aestivum L.) cDNA clone encoding the waxy protein. Plant Mol Biol 16:1099–1101CrossRefPubMedGoogle Scholar
  11. Costea M, Sanders A, Waines G (2001) Preliminary results toward a revision of the Amaranthus hybridus species complex (Amaranthaceae). Sida 19:931–974Google Scholar
  12. Costea M, Brenner DM, Tardif FJ, Tan YF, Sun M (2006) Delimitation of Amaranthus cruentus L. and Amaranthus caudatus L. using micromorphology and AFLP analysis: an application in germplasm identification. Genet Resour Crop Evol 53:1625–1633CrossRefGoogle Scholar
  13. Davis J, Supatcharee N, Khandellwal R, Chibbar R (2003) Synthesis of novel starches in Planta: opportunities and challenges. Starch/Staerke 55:107–120CrossRefGoogle Scholar
  14. Domon E, Fujita M, Ishikawa N (2002a) The insertion/deletion polymorphisms in the waxy gene of barley genetic resources from East Asia. Theor Appl Genet 104:132–138CrossRefPubMedGoogle Scholar
  15. 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 77:351–359CrossRefPubMedGoogle Scholar
  16. Dry I, Smith A, Edward A, Bhattacharyya M, Dunn P, Martin C (1992) Characterization of cDNAs encoding two isoforms of granule-bound starch synthase which show differential expression in developing storage organs of pea and potato. Plant J 2:193–202PubMedGoogle Scholar
  17. Echt C, 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
  18. Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978CrossRefPubMedGoogle Scholar
  19. Fedoroff N, Wessler S, Shure M (1983) Isolation of the transposable maize controlling elements Ac and Ds. Cell 35:235–242CrossRefPubMedGoogle Scholar
  20. 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 268:214–222CrossRefPubMedGoogle Scholar
  21. Furukawa K, Tagaya M, Inouye M, Preiss J, Fukui T (1990) Identification of lysine 15 at the active site in Escherichia coli glycogen synthase. Conservation of Lys-X-Gly-Gly sequence in the bacterial and mammalian enzymes. J Biol Chem 265:2086–2090PubMedGoogle Scholar
  22. Hirano HY, Eiguchi M, Sano Y (1998) A single base change altered the regulation of the waxy gene at the posttranscriptional level during the domestication of rice. Mol Biol Evol 15:978–987PubMedGoogle Scholar
  23. Hovenkamp-Hermelink J, Jacobsen E, Ponstein A, Visser R, Vos-Scheperkeuter G, Bijmolt E, Vries J, Witholt B, Feenstra W (1987) Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.). Theor Appl Genet 75:217–221CrossRefGoogle Scholar
  24. Hseih J (1988) Genetic studies of the Wx gene of sorghum (Sorghum bicolor [L.] Moench). Bot Bull Academia Sinica 29:293–299Google Scholar
  25. Hsieh J, Liu C, Hsing Y (1996) Molecular cloning of a sorghum cDNA encoding the seed waxy protein. Plant Physiol 112:1735CrossRefGoogle Scholar
  26. Inouchi N, Nishi K, Tanaka S, Asai M, Kawase Y, Hata Y, Konishi Y, Yue S, Fuwa H (1999) Characterization of amaranth and quinoa starches. J Appl Glycosci 46:233–240Google Scholar
  27. Isshiki M, Yamamoto Y, Satoh H, Shimamoto K (2001) Nonsense-mediated decay of mutant waxy mRNA in rice. Plant Physiol 125:1388–1395CrossRefPubMedGoogle Scholar
  28. Juan R, Pastor J, Alaiz M, Vioque J (2007) Electrophoretic characterization of Amaranthus L. seed proteins and its systematic implications. Bot J Linn Soc 155:57–63CrossRefGoogle Scholar
  29. Kawase M, Fukunaga K, Kato K (2005) Diverse origins of waxy foxtail millet crops in East and Southeast Asia mediated by multiple transposable element insertions. Mol Genet Genomics 274:131–140CrossRefPubMedGoogle Scholar
  30. Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120CrossRefPubMedGoogle Scholar
  31. Kimura T, Ideta O, Saito A (2000) Identification of the gene encoding granule-bound starch synthase I in sweet potato (Ipomoea batatas (L.) Lam.). Plant Biotechnol 17:247–252Google Scholar
  32. Klosgen R, Gierl A, Schwarz-Sommer Z, Saedler H (1986) Molecular analysis of the waxy locus of Zea mays. Mol Genet Genomics 203:237–244CrossRefGoogle Scholar
  33. Konishi Y, Nojima H, Okuno K, Asaoka M, Fuwa H (1985) Characterization of starch granules from waxy, nonwaxy, and hybrid seeds of Amaranthus hypochondriacus L. Agri Biol Chem 49:1965–1971Google Scholar
  34. Kumar A, Larsen C, Preiss J (1986) Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli ADP-glucose: alpha-1, 4-glucan, 4-glucosyltransferase as deduced from the nucleotide sequence of the glgA gene. J Biol Chem 261:16256–16259PubMedGoogle Scholar
  35. Lanoue KZ, Wolf PG, Browning S, Hood EE (1996) Phylogenetic analysis of restriction-site variation in wild and cultivated Amaranthus species (Amaranthaceae). Theor Appl Genet 93:722–732CrossRefGoogle Scholar
  36. Marcone M (2001) Starch properties of Amaranthus pumilus (seabeach amaranth): a threatened plant species with potential benefits for the breeding/amelioration of present Amaranthus cultivars. Food Chem 73:61–66CrossRefGoogle Scholar
  37. Mochizuki K, Umeda M, Ohtsubo H, Ohtsubo E (1992) Characterization of a plant SINE, p-SINE1, in rice genomes. Jpn J Genet 67:155–166CrossRefPubMedGoogle Scholar
  38. Murai J, Taira T, Ohta D (1999) Isolation and characterization of the three waxy genes encoding the granule-bound starch synthase in hexaploid wheat. Gene 234:71–79CrossRefPubMedGoogle Scholar
  39. Murray M, Thompson W (1980) Rapid isolation of high molecular weight plant DNA. Nucl Acids Res 8:4321–4325Google Scholar
  40. Nakamura T, Vrinten P, Hayakawa K, Ikeda J (1998) Characterization of a granule-bound starch synthase isoform found in the pericarp of wheat. Plant Physiol 118:451–459CrossRefPubMedGoogle Scholar
  41. Nakayama H, Afzal M, Okuno K (1998) Intraspecific differentiation and geographical distribution of Wx alleles for low amylose content in endosperm of foxtail millet, Setaria italica (L.) Beauv. Euphytica 102:289–293CrossRefGoogle Scholar
  42. Nelson O, Rines H (1962) The enzymatic deficiency in the waxy mutant of maize. Biochem Biophys Res Commun 9:297–300CrossRefPubMedGoogle Scholar
  43. Okagaki R (1992) Nucleotide sequence of a long cDNA from the rice waxy gene. Plant Mol Biol 19:513–516CrossRefPubMedGoogle Scholar
  44. Okuno K, Sakaguchi S (1981) Glutinous and non-glutinous starches in perisperm of grain amaranths. Cereal Res Commun 9:305–310Google Scholar
  45. Okuno K, Sakaguchi S (1982) Inheritance of starch characteristics in perisperm of Amaranthus hypochondriacus. J Hered 73:467Google Scholar
  46. Park YJ, Nemoto K, Nishikawa T, Matsushima K, Minami M, Kawase M (2009) Molecular cloning and characterization of granule bound starch synthase I cDNA from a grain amaranth (Amaranthus cruentus L.). Breed Sci (in press)Google Scholar
  47. Rohde W, Becker D, Salamini F (1988) Structural analysis of the waxy locus from Hordeum vulgare. Nucl Acids Res 16:7185CrossRefPubMedGoogle Scholar
  48. Salehuzzaman S, Jacobsen E, Visser R (1993) Isolation and characterization of a cDNA encoding granule-bound starch synthase in cassava (Manihot esculenta Crantz) and its antisense expression in potato. Plant Mol Biol 23:947–962CrossRefPubMedGoogle Scholar
  49. Sano Y (1984) Differential regulation of waxy gene expression in rice endosperm. Theor Appl Genet 68:467–473CrossRefGoogle Scholar
  50. Sauer JD (1950) The grain amaranths: a survey of their history and classification. Ann Missouri Bot Gard 37:561–619CrossRefGoogle Scholar
  51. Sauer JD (1967) The grain amaranths and their relatives: a revised taxonomic and geographic survey. Ann Missouri Bot Gard 54:103–137CrossRefGoogle Scholar
  52. Saunders R, Becker R (1984) Amaranthus: a potential food and feed resource. Adv Cereal Sci Technol 6:357–396Google Scholar
  53. Sprague G, Brimhall B, Hixon R (1943) Some effects of the waxy gene in corn on properties of the endosperm starch. J Am Soc Agron 35:817Google Scholar
  54. Sugimoto Y, Yamada K, Sakamoto S, Fuwa H (1981) Some properties of normal-and waxy-type starches of Amaranthus hypochondriacus L. pp 112–116Google Scholar
  55. Swofford D (1988) PAUP*: phylogenetic analysis using parsimony and other methods, version 4.0 (test ver. 61–64). Sinauer Associates Publishers, SunderlandGoogle Scholar
  56. Thompson JDG, Plewniak FJ, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882CrossRefGoogle Scholar
  57. Tsai C (1974) The function of the waxy locus in starch synthesis in maize endosperm. Biochem Genetics 11:83–96CrossRefGoogle Scholar
  58. van der Leij F, Visser R, Ponstein A, Jacobsen E, Feenstra W (1991) Sequence of the structural gene for granule-bound starch synthase of potato (Solanum tuberosum L.) and evidence for a single point deletion in the amf allele. Mol Gen Genet 228:240–248CrossRefPubMedGoogle Scholar
  59. Van K, Onoda S, Kim MY, Kim KD, Lee SH (2008) Allelic variation of the Waxy gene in foxtail millet [Setaria italica (L.) P. Beauv.] by single nucleotide polymorphisms. Mol Genet Genom 279:255–266CrossRefGoogle Scholar
  60. Vrinten P, Nakamura T, Yamamori M (1999) Molecular characterization of waxy mutations in wheat. Mol Genet Genomics 261:463–471CrossRefGoogle Scholar
  61. Wang Z, Wu Z, Xing Y, Zheng F, Guo X, Zhang W, Hong M (1990) Nucleotide sequence of rice waxy gene. Nucl Acids Res 18:5898CrossRefPubMedGoogle Scholar
  62. Wang Z, Zheng F, Shen G, Gao J, Snustad D, Li M, Zhang J, Hong M (1995) The amylose content in rice endosperm is related to the post-transcriptional regulation of the waxy gene. Plant J 7:613–622CrossRefPubMedGoogle Scholar
  63. Wessler S, Varagona M (1985) Molecular basis of mutations at the waxy locus of maize: correlation with the fine structure genetic map. Proc Natl Acad Sci 82:4177–4181CrossRefPubMedGoogle Scholar
  64. Xu F, Sun M (2001) Comparative analysis of phylogenetic relationships of grain amaranths and their wild relatives (Amaranthus; Amaranthaceae) using internal transcribedspacer, amplified fragment length polymorphism, and double-primer fluorescent intersimple sequence repeat markers. Mol Phylogenet Evol 21:372–387CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Young-Jun Park
    • 1
  • Kazuhiro Nemoto
    • 2
  • Tomotaro Nishikawa
    • 3
  • Kenichi Matsushima
    • 2
  • Mineo Minami
    • 2
  • Makoto Kawase
    • 3
  1. 1.Department of Bioscience and Food Production Science, Interdisciplinary Graduate School of Science & TechnologyShinshu UniversityNaganoJapan
  2. 2.Graduate School of AgricultureShinshu UniversityNaganoJapan
  3. 3.Genebank, National Institute of Agrobiological SciencesIbarakiJapan

Personalised recommendations