Functional & Integrative Genomics

, Volume 12, Issue 1, pp 131–141 | Cite as

Mutagenesis of barley malting quality QTLs with Ds transposons

  • Surinder Singh
  • Han Qi Tan
  • Jaswinder SinghEmail author
Original Paper


Various functional genomic tools are being used to identify and characterize genes in plants. The Activator/Dissociation (Ac/Ds) transposon-based approach offers great potential, especially in barley, due to its limited success of genetic transformation and its large genome size. The bias of the Ac/Ds system towards genic regions and its tendency toward localized transpositions can greatly enhance the discovery and tagging of genes linked to Ds. Barley is a key ingredient in malting and brewing industry; therefore, gene discovery in relation to malting has an industrial perspective. Malting quality in barley is a complex and quantitatively inherited trait. Two major quantitative trait loci (QTLs) affecting malting quality traits have been located on chromosome 4H. In this study, Ds was reactivated from parent transposants (TNP) lines, TNP-29 and TNP-79, where Ds was mapped in the vicinity of important malting QTLs. Reactivation of Ds was carried out both by conventional breeding and in vitro approaches. A threefold increase in reactivation frequency through the in vitro approach enabled the development of a new genomic resource for the dissection of malting QTL and gene discovery in barley. Identification of unique flanking sequences, using high-efficiency thermal asymmetric interlaced PCR and inverse PCR from these populations, has further emphasized the new location of Ds in the barley genome and provided new transposon mutants especially in β-GAL1, β-amylase-like gene and ABC transporter for functional genomic studies.


Ac/Ds Transposon reactivation Genomics Transformation Barley Cereals 



This work was supported by the Brewing and Malting Barley Research Institute and the Natural Sciences and Engineering Research Council of Canada Collaborative Research and Development. We also thank Dr. Ravneet Kaur and Dr. Manjit Singh for critically reading this manuscript. The authors also thank Mr. Neil Dylan Lamb-Palmer for his suggestions about bioinformatic analysis.


  1. Alonso JM, Ecker JR (2006) Moving forward in reverse: genetic technologies to enable genome-wide phenomic screens in Arabidopsis. Nat Rev Genet 7:524–536PubMedCrossRefGoogle Scholar
  2. Arumuganathan K, Earle E (1991) Nuclear DNA content of some important plant species. Plant Mol Biol Rep 9:208–218CrossRefGoogle Scholar
  3. Ayliffe MA, Agostino A, Clarke BC, Furbank R, von Caemmerer S, Pryor AJ (2009) Suppression of the barley uroporphyrinogen III synthase gene by a Ds activation tagging element generates developmental photosensitivity. Plant Cell 21:814–831PubMedCrossRefGoogle Scholar
  4. Baker B, Schell J, Jeff S, Horst L, Nina F (1986) Transposition of the maize controlling element Activator in tobacco. Proc Natl Acad Sci U S A 83:4844–4848PubMedCrossRefGoogle Scholar
  5. Bennetzen JL, Ma J, Devos KM (2005) Mechanisms of recent genome size variation in flowering plants. Ann Bot 95:127–132PubMedCrossRefGoogle Scholar
  6. Brettell RIS, Dennis ES (1991) Reactivation of a silent Ac following tissue culture is associated with heritable alterations in its methylation pattern. Mol Gen Genet 229:365–372PubMedCrossRefGoogle Scholar
  7. Caldwell DG, McCallum N, Shaw P, Muehlbauer GJ, Marshall DF, Waugh R (2004) A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.). Plant J 40:143PubMedCrossRefGoogle Scholar
  8. Chin H, Choe M, Lee S, Park S, Koo J, Kim N, Lee J, Oh B, Yi G, Kim S, Choi H, Cho M, Han C (1999) Molecular analysis of rice plants harboring an Ac/Ds transposable element mediated gene trapping system. Plant J 19:615–623PubMedCrossRefGoogle Scholar
  9. Chul Min K, Byoung J II, Hai Long P, Soon Ju P, Min Jung K, Sung Han P, Jin Young P, Su Hyun P, Eun Kyeong L, Nam Soo C, Yong Jae W, Gi Hwan L, Min Hee N, Doh Won Y, Myung Chul L, Young Soon C, Kon Ho L, Moo Young E, Chang-deok H (2002) Reprogramming of the activity of activator/dissociation transposon family during plant regeneration in rice. Mol Cells 14:231–237Google Scholar
  10. Close TJ, Wanamaker SI, Caldo RA, Turner SM, Ashlock DA, Dickerson JA, Wing RA, Muehlbauer GJ, Kleinhofs A, Wise RP (2004) A new resource for cereal genomics: 22K barley GeneChip comes of age. Plant Physiol 134:960–968PubMedCrossRefGoogle Scholar
  11. Close TJ, Bhat PR, Lonardi S, Wu YH, Rostoks N, Ramsay L, Druka A, Stein N, Svensson JT, Wanamaker S, Bozdag S, Roose ML, Moscou MJ, Chao SAM, Varshney RK, Szucs P, Sato K, Hayes PM, Matthews DE, Kleinhofs A, Muehlbauer GJ, DeYoung J, Marshall DF, Madishetty K, Fenton RD, Condamine P, Graner A, Waugh R (2009) Development and implementation of high-throughput SNP genotyping in barley. BMC Genomics 10:13CrossRefGoogle Scholar
  12. Colasanti J, Yuan Z, Sundaresan V (1998) The indeterminate gene encodes a zinc finger protein and regulates a leaf-generated signal required for the transition to flowering in maize. Cell 93:593–603PubMedCrossRefGoogle Scholar
  13. Cooper L, Marquez-Cedillo L, Singh JS, Sturbaum AK, Zhang S, Edwards V, Johnson K, Kleinhofs A, Rangel S, Carollo V, Bregitzer P, Lemaux PG, Hayes PM (2004) Mapping Ds insertions in barley using a sequence-based approach. Mol Genet Genomics 272:181–193PubMedCrossRefGoogle Scholar
  14. Coupland G, Baker B, Schell J, Starlinger P (1988) Characterization of the maize transposable element-Ac by internal deletions. EMBO J 7:3653–3659PubMedGoogle Scholar
  15. Cowperthwaite M, Park W, Xu Z, Yan X, Maurais SC, Dooner HK (2002) Use of the transposon Ac as a gene-searching engine in the maize genome. Plant Cell 14:713–726PubMedCrossRefGoogle Scholar
  16. DeLong A, Calderonurrea A, Dellaporta SL (1993) Sex determination gene tasselseed2 of maize encodes a short-chain alcohol-dehydrogenase required for stage-specific floral organ abortion. Cell 74:757–768PubMedCrossRefGoogle Scholar
  17. Gao W, Clancy JA, Han F, Jones BL, Budde A, Wesenberg DM, Kleinhofs A, Ullrich SE (2004) Fine mapping of a malting-quality QTL complex near the chromosome 4H S telomere in barley. Theor Appl Genet 109:750–760PubMedCrossRefGoogle Scholar
  18. Gill BS, Appels R, Botha-Oberholster A-M, Buell CR, Bennetzen JL, Chalhoub B, Chumley F, Dvorak J, Iwanaga M, Keller B, Li W, McCombie WR, Ogihara Y, Quetier F, Sasaki T (2004) A workshop report on wheat genome sequencing: International Genome Research on Wheat Consortium. Genetics 168:1087–1096PubMedCrossRefGoogle Scholar
  19. Greco R, Ouwerkerk PBF, de Kam RJ, Sallaud C, Favalli C, Colombo L, Guiderdoni E, Meijer AH, Hoge JHC, Pereira A (2003) Transpositional behaviour of an Ac/Ds system for reverse genetics in rice. Theor Appl Genet 108:10–24PubMedCrossRefGoogle Scholar
  20. Gurel S, Gurel E, Kaur R, Wong J, Meng L, Tan H-Q, Lemaux P (2009) Efficient reproducible Agrobacterium-mediated transformation of sorghum using heat treatment of immature embryos. Plant Cell Rep 28:429–444PubMedCrossRefGoogle Scholar
  21. Hayes PM, Liu BH, Knapp SJ, Chen F, Jones B, Blake T, Franckowiak J, Rasmusson D, Sorrells M, Ullrich SE, Wesenberg D, Kleinhofs A (1993) Quantitative trait locus effects and environmental interaction in a sample of North-American barley germplasm. Theor Appl Genet 87:392–401CrossRefGoogle Scholar
  22. Ito T, Motohashi R, Kuromori T, Mizukado S, Sakurai T, Kanahara H, Seki M, Shinozaki K (2002) A new resource of locally transposed dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome. Plant Physiol 129:1695–1699PubMedCrossRefGoogle Scholar
  23. Izawa T, Ohnishi T, Nakano T, Ishida N, Enoki H, Hashimoto H, Itoh K, Terada R, Wu C, Miyazaki C, Endo T, Iida S, Shimamoto K (1997) Transposon tagging in rice. Plant Mol Biol 35:219–229PubMedCrossRefGoogle Scholar
  24. Jiang SY, Ramachandran S (2010) Natural and artificial mutants as valuable resources for functional genomics and molecular breeding. Int J Biol Sci 6:228–251PubMedCrossRefGoogle Scholar
  25. Keller B, Feuillet C (2000) Colinearity and gene density in grass genomes. Trends Plant Sci 5:246–251PubMedCrossRefGoogle Scholar
  26. Keller B, Feuillet C, Yahiaoui N (2005) Map-based isolation of disease resistance genes from bread wheat: cloning in a supersize genome. Genet Res 85:93–100PubMedCrossRefGoogle Scholar
  27. Kohli A, Prynne MQ, Miro B, Pereira A, Twyman RM, Capell T, Christou P (2004) Dedifferentiation-mediated changes in transposition behavior make the Activator; transposon an ideal tool for functional genomics in rice. Mol Breed 13:177–191CrossRefGoogle Scholar
  28. Kolesnik T, Szeverenyi I, Bachmann D, Kumar CS, Jiang S, Ramamoorthy R, Cai M, Ma ZG, Sundaresan V, Ramachandran S (2004) Establishing an efficient Ac/Ds tagging system in rice: large-scale analysis of Ds flanking sequences. Plant J 37:301–314PubMedCrossRefGoogle Scholar
  29. Koprek T, McElroy D, Louwerse J, Williams-Carrier R, Lemaux PG (2000) An efficient method for dispersing Ds elements in the barley genome as a tool for determining gene function. Plant J 24:253–263PubMedCrossRefGoogle Scholar
  30. Kunze R, Starlinger P (1989) The putative transposase of transposable element Ac from Zea mays L. interacts with subterminal sequences of Ac. EMBO J 8:3177–3185PubMedGoogle Scholar
  31. Kuromori T, Hirayama T, Kiyosue Y, Takabe H, Mizukado S, Sakurai T, Akiyama K, Kamiya A, Ito T, Shinozaki K (2004) A collection of 11 800 single-copy Ds transposon insertion lines in Arabidopsis. Plant J 37:897–905PubMedCrossRefGoogle Scholar
  32. Lee G-J, Wu X, Shannon JG, Sleper DA, Nguyen HT (2007) Soyabean. In: Kole C (ed) Genome wide and molecular breeding in plants-Oliseeds. Springer, Heidelberg, pp 1–45Google Scholar
  33. Li J, Jiang D, Zhou H, Li F, Yang J, Hong L, Fu X, Li Z, Liu Z, Li J, Zhuang C (2011) Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice. PLoS One 6:e17444BreGoogle Scholar
  34. Marsch-Martínez N, Pereira A (2011) Activation Tagging with En/Spm-I/dSpm Transposons in Arabidopsis. In: Pereira A (ed) Methods in Molecular Biology, vol 678. Springer, New York, pp 91–105Google Scholar
  35. McElroy D, Louwerse JD, McElroy SM, Lemaux PG (1997) Development of a simple transient assay for Ac/Ds activity in cells of intact barley tissue. Plant J 11:157–165PubMedCrossRefGoogle Scholar
  36. Nakagawa Y, Machida C, Machida Y, Toriyama K (2000) Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants. Plant Cell Physiol 41:733–742PubMedGoogle Scholar
  37. Parinov S, Sevugan M, Ye D, Yang W-C, Kumaran M, Sundaresan V (1999) Analysis of flanking sequences from dissociation insertion lines: a database for reverse genetics in Arabidopsis. Plant Cell 11:2263–2270PubMedCrossRefGoogle Scholar
  38. Peters JL, Cnudde F, Gerats T (2003) Forward genetics and map-based cloning approaches. Trends Plant Sci 8:484–491PubMedCrossRefGoogle Scholar
  39. Potokina E, Caspers M, Prasad M, Kota R, Zhang H, Sreenivasulu N, Wang GM (2004) Functional association between malting quality trait components and cDNA array based expression patterns in barley (Hordeum vulgare L.). Mol Breed 14:153–170CrossRefGoogle Scholar
  40. Potokina E, Prasad M, Kota R, Zhang H, Sreenivasulu N, Wang GM (2006) Expression genetics and haplotype analysis reveal cis regulation of serine carboxypeptidase I (Cxp1), a candidate gene for malting quality in barley (Hordeum vulgare L.). Funct Intr Genomics 6:25–35CrossRefGoogle Scholar
  41. Qu S, Jeon J-S, Ouwerkerk PBF, Bellizzi M, Leach J, Ronald P, Wang G-L (2009) Construction and application of efficient Ac–Ds transposon tagging vectors in rice. J Integr Plant Bio 51:982–992CrossRefGoogle Scholar
  42. Randhawa HS, Singh J, Lemaux PG, Gill KS (2009) Mapping barley Ds insertions using wheat deletion lines reveals high insertion frequencies in gene-rich regions with high to moderate recombination rates. Genome 52:566–575PubMedCrossRefGoogle Scholar
  43. Ros F, Kunze R (2001) Regulation of activator/dissociation transposition by replication and DNA methylation. Genetics 157:1723–1733PubMedGoogle Scholar
  44. Sato K, Close TJ, Bhat P, Munoz-Amatriain M, Muehlbauer GJ (2011) Single nucleotide polymorphism mapping and alignment of recombinant chromosome substitution lines in barley. Plant Cell Physiol 52:728–737PubMedCrossRefGoogle Scholar
  45. Schulte D, Close TJ, Graner A, Langridge P, Matsumoto T, Muehlbauer G, Sato K, Schulman AH, Waugh R, Wise RP, Stein N (2009) The international barley sequencing consortium: at the threshold of efficient access to the barley genome. Plant Physiol 149:142–147PubMedCrossRefGoogle Scholar
  46. Singh M, Lewis PE, Hardeman K, Bai L, Rose JKC, Mazourek M, Chomet P, Brutnell TP (2003) Activator mutagenesis of the Pink scutellum1/viviparous7 locus of maize. Plant Cell 15:874–884PubMedCrossRefGoogle Scholar
  47. Singh J, Zhang S, Chen C, Cooper L, Bregitzer P, Sturbaum A, Hayes P, Lemaux P (2006) High-frequency Ds remobilization over multiple generations in barley facilitates gene tagging in large genome cereals. Plant Mol Biol 62:937–950PubMedCrossRefGoogle Scholar
  48. Singh J, Freeling M, Lisch D (2008) A position effect on the heritability of epigenetic silencing. PLOS Gen 4:e1000216CrossRefGoogle Scholar
  49. Smith D, Yanai Y, Liu YG, Ishiguro S, Okada K, Shibata D, Whittier RF, Fedoroff NV (1996) Characterization and mapping of Ds-GUS-T-DNA lines for targeted insertional mutagenesis. Plant J 10:721–732PubMedCrossRefGoogle Scholar
  50. Smulders MJM, De Klerk GJ (2011) Epigenetics in plant tissue culture. Plant Growth Regul 63:137–146CrossRefGoogle Scholar
  51. Sreenivasulu N, Graner A, Wobus U (2008) Barley genomics: an overview. Int J Plant Gen 2008:1–13CrossRefGoogle Scholar
  52. Talamè V, Bovina R, Sanguineti MC, Tuberosa R, Lundqvist U, Salvi S (2008) TILLMore, a resource for the discovery of chemically induced mutants in barley. Plant Biotech J 6:477–485CrossRefGoogle Scholar
  53. Tan HQ, Singh J (2011) High-efficiency thermal asymmetric interlaced (HE-TAIL) PCR for amplification of Ds transposon insertion sites in barley. J Plant Mol Biol Biotechnol 2:9–14Google Scholar
  54. Triantafillidou D, Georgatsos JG (2001) Barley β-Galactosidase: structure, function, heterogeneity, and gene origin. J Protein Chem 7:551–562CrossRefGoogle Scholar
  55. Upadhyaya N, Zhou X-R, Zhu Q-H, Ramm K, Wu L, Eamens A, Sivakumar R, Kato T, Yun D-W, Santhoshkumar C, Narayanan K, Peacock J, Dennis E (2002) An Ac/Ds gene and enhancer trapping system for insertional mutagenesis in rice. Funct Plant Biol 29:547–559CrossRefGoogle Scholar
  56. Upadhyaya N, Zhu QH, Zhou XR, Eamens AL, Hoque MS, Ramm K, Shivakkumar R, Smith KF, Pan ST, Li S, Peng K, Kim SJ, Dennis ES (2006) Dissociation (Ds) constructs mapped Ds launch pads and a transiently-expressed transposase system suitable for localized insertional mutagenesis in rice. Theor Appl Genet 112:1326–1341PubMedCrossRefGoogle Scholar
  57. Varagona MJ, Purugganan M, Wessler SR (1990) Alternative splicing induced by insertion of retrotransposons into the maize waxy gene. Plant Cell 4:811–820CrossRefGoogle Scholar
  58. Vinje M, Willis D, Duke S, Henson C (2011) Differential expression of two β-amylase genes (Bmy1 and Bmy2) in developing and mature barley grain. Planta 233:1001–1010PubMedCrossRefGoogle Scholar
  59. Watanabe S, Hideshima R, Xia Z, Tsubokura Y, Sato S, Nakamoto Y, Yamanaka N, Takahashi R, Ishimoto M, Anai T, Tabata S, Harada K (2009) Map-based cloning of the gene associated with the soybean maturity locus E3. Genetics 182:1251–1262PubMedCrossRefGoogle Scholar
  60. Watanabe S, Xia Z, Hideshima R, Tsubokura Y, Sato S, Yamanaka N, Takahashi R, Anai T, Tabata S, Kitamura K, Harada K (2011) A map-based cloning strategy employing a residual heterozygous line reveals that the gigantea gene is involved in soybean maturity and flowering. Genetics 188:395–407PubMedCrossRefGoogle Scholar
  61. Watson L, Henry R (2005) Microarray analysis of gene expression in germinating barley embryos. (Hordeum vulgare L.). Funct Integr Genomics 5:155–162PubMedCrossRefGoogle Scholar
  62. Wei K, Xue D-W, Huang Y-Z, Jin X-I, Wu F-B, Zhang G-P (2009) Genetic mapping of quantitative trait loci associated with β-amylase and limit dextrinase activities and β-glucan and protein fraction contents in barley. J Zhejiang Univ Sci B 10:839–846PubMedCrossRefGoogle Scholar
  63. Weld RJ, Bicknell RA, Heinemann JA, Eady CC (2002) Ds transposition mediated by transient transposase expression in Heiracium aurantiacum. Plant Cell Tiss Organ Cul 69:45–54CrossRefGoogle Scholar
  64. White J, Pacey-Miller T, Bundock P, Henry R (2008) Differential LongSAGE tag abundance analysis in a barley seed germination time course and validation with relative real-time RT-PCR. Plant Sci 175:858–867CrossRefGoogle Scholar
  65. Zale JM, Clancy JA, Ullrich SE, Jones BL, Hayes PM (2000) North American barley genome mapping project. Summary of barley malting quality QTL mapped in various populations. Barley Genet Newslett 30:44Google Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Plant Science DepartmentMcGill UniversityQuebecCanada

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