Resources for Reverse Genetics Approaches in Brassica Species

  • Thomas WoodEmail author
  • Pauline Stephenson
  • Lars Østergaard
Part of the Plant Genetics and Genomics: Crops and Models book series (PGG, volume 9)


Gene, genomic and genome sequences are being generated with incredible speed, thanks to the advent of cheaper and faster sequencing technologies. For plants, the Arabidopsis thaliana (Col-0) genome was sequenced in its entirety in 2000, and a number of whole-genome sequences in additional A. thaliana ecotypes have been completed since then, providing an amazing resource for functional genomics in this species. Although Arabidopsis is still the only genus of the Brassicaceae family to have its genome completely sequenced, a multinational effort is currently ongoing to obtain first the sequence of the A genome (Brassica rapa) and ultimately all the cultivated Brassicas of the “U triangle”. The obvious challenge is therefore what to do with this massive amount of information. How does these data expand our knowledge of plant biology or aid in the development of tools for crop improvement? In this chapter we will describe the current status of the principal resources available for reverse genetics approaches in the Brassica genus, TILLING and RNAi, and discuss their advantages and disadvantages for the study of plant biology and the development of tools for crop improvement.


Reverse genetics Gene silencing TILLING Gene function 


  1. Alonso JM, Stepanova AN (2003) T-DNA mutagenesis in Arabidopsis. Methods Mol Biol 236:177–188PubMedGoogle Scholar
  2. Alvarez JP, Pekker I, Goldshmidt A et al (2006) Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species. Plant Cell 18:1134–1151CrossRefPubMedGoogle Scholar
  3. Bartlett JG, Alves SC, Smedley M et al (2008) High-throughput Agrobacterium-mediated barley transformation. Plant Methods 4:22CrossRefPubMedGoogle Scholar
  4. Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363CrossRefPubMedGoogle Scholar
  5. Baumberger N, Baulcombe DC (2005) Arabidopsis ARGONAUTE 1 is an RNA slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci USA 102:11928–11933CrossRefPubMedGoogle Scholar
  6. Blanc G, Wolfe KH (2004a) Functional divergence of duplicated genes formed by polyploidy during Arabidopsis evolution. Plant Cell 16:1679–1691CrossRefPubMedGoogle Scholar
  7. Blanc G, Wolfe KH (2004b) Widespread paleopolyploidy in model plant species inferred from age distributions of duplicate genes. Plant Cell 16:1667–1678CrossRefPubMedGoogle Scholar
  8. Bleys A, Van Houdt H, Depicker A (2006a) Down-regulation of endogenes mediated by a transitive silencing signal. Rna-a Publication of the Rna Society 12:1633–1639Google Scholar
  9. Bleys A, Vermeersch L, Van Houdt H et al (2006b) The frequency and efficiency of endogene suppression by transitive silencing signals is influenced by the length of sequence homology. Plant Physiol 142:788–796CrossRefPubMedGoogle Scholar
  10. Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M et al (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320:1185–1190CrossRefPubMedGoogle Scholar
  11. Brodersen P, Voinnet O (2006) The diversity of RNA silencing pathways in plants. Trends Genet 22:268–280CrossRefPubMedGoogle Scholar
  12. Burch-Smith TM, Anderson JC, Martin GB et al (2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J 39:734–746CrossRefPubMedGoogle Scholar
  13. Burch-Smith TM, Schiff M, Liu Y et al (2006) Efficient virus-induced gene silencing in Arabidopsis. Plant Physiol 142:21–27CrossRefPubMedGoogle Scholar
  14. Byzova M, Verduyn C, De Brouwer D et al (2004) Transforming petals into sepaloid organs in Arabidopsis and oilseed rape: implementation of the hairpin RNA-mediated gene silencing technology in an organ-specific manner. Planta 218:379–387CrossRefPubMedGoogle Scholar
  15. Caldwell DG, McCallum N, Shaw P et al (2004) A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.). Plant J 40:143–150CrossRefPubMedGoogle Scholar
  16. Carmell MA, Xuan Z, Zhang MQ et al (2002) The Argonaute family:tentacles that reach into RNAi, developmental control, stem cell maintainance, and tumorigenesis. Genes Dev 16:2733–2742CrossRefPubMedGoogle Scholar
  17. Chuang CF, Meyerowitz EM (2000) Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci USA 97:4985–4990CrossRefPubMedGoogle Scholar
  18. Cooper JL, Till BJ, Laport RG et al (2008) TILLING to detect induced mutations in soybean. BMC Plant Biol 8:9CrossRefPubMedGoogle Scholar
  19. Craft J, Samalova M, Baroux C et al (2005) New pOp/LhG4 vectors for stringent glucocorticoid-dependent transgene expression in Arabidopsis. Plant J 41:899–918CrossRefPubMedGoogle Scholar
  20. Dalmais M, Schmidt J, Le Signor C et al (2008) UTILLdb, a Pisum sativum in silico forward and reverse genetics tool. Genome Biol 9:R43CrossRefPubMedGoogle Scholar
  21. Dalmay T, Hamilton A, Rudd S et al (2000) An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101:543–553CrossRefPubMedGoogle Scholar
  22. Duan CG, Wang CH, Fang RX et al (2008) Artificial MicroRNAs highly accessible to targets confer efficient virus resistance in plants. J Virol 82:11084–11095CrossRefPubMedGoogle Scholar
  23. Eamens A, Wang MB, Smith NA et al (2008) RNA silencing in plants: yesterday, today, and tomorrow. Plant Physiol 147:456–468CrossRefPubMedGoogle Scholar
  24. Eason JR, Ryan DJ, Watson LM et al (2005) Suppression of the cysteine protease, aleurain, delays floret senescence in Brassica oleracea. Plant Mol Biol 57:645–657CrossRefPubMedGoogle Scholar
  25. Ecker JR, Davis RW (1986) Inhibition of gene expression in plant cells by expression of antisense RNA. Proc Natl Acad Sci USA 83:5372–5376CrossRefPubMedGoogle Scholar
  26. Fire A, Xu S, Montgomery MK et al (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811CrossRefPubMedGoogle Scholar
  27. Franco-Zorrilla JM, Valli A, Todesco M et al (2007) Target mimicry provides a new mechanism for regulation of microRNA activity. Nat Genet 39:1033–1037CrossRefPubMedGoogle Scholar
  28. Fusaro AF, Matthew L, Smith NA et al (2006) RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway. EMBO Rep 7:1168–1175CrossRefPubMedGoogle Scholar
  29. Gilchrist EJ, O’Neil NJ, Rose AM et al (2006) TILLING is an effective reverse genetics technique for Caenorhabditis elegans. BMC Genomics 7:262CrossRefPubMedGoogle Scholar
  30. Greene EA, Codomo CA, Taylor NE et al (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164:731–740PubMedGoogle Scholar
  31. Hamilton A, Voinnet O, Chappell L et al (2002) Two classes of short interfering RNA in RNA silencing. Embo J 21:4671–4679CrossRefPubMedGoogle Scholar
  32. Hammond SM, Caudy AA, Hannon GJ (2001) Post-transcriptional gene silencing by double-stranded RNA. Nat Rev Genet 2:110–119CrossRefPubMedGoogle Scholar
  33. Helliwell C, Waterhouse P (2003) Constructs and methods for high-throughput gene silencing in plants. Methods 30:289–295CrossRefPubMedGoogle Scholar
  34. Helliwell CA, Waterhouse PM (2005) Constructs and methods for hairpin RNA-mediated gene silencing in plants. Methods Enzymol 392:24–35CrossRefPubMedGoogle Scholar
  35. Helliwell CA, Wesley SV, Wielopolska AJ et al (2002) High-throughput vectors for efficient gene silencing in plants. Funct Plant Biol 29:1217–1225CrossRefGoogle Scholar
  36. Henikoff S, Till BJ, Comai L (2004) TILLING. Traditional mutagenesis meets functional genomics. Plant Physiol 135:630–636CrossRefPubMedGoogle Scholar
  37. Hirai S, Oka S, Adachi E et al (2007) The effects of spacer sequences on silencing efficiency of plant RNAi vectors. Plant Cell Rep 26:651–659CrossRefPubMedGoogle Scholar
  38. Horiguchi G (2004) RNA silencing in plants: a shortcut to functional analysis. Differentiation 72:65–73CrossRefPubMedGoogle Scholar
  39. Jadhav A, Katavic V, Marillia EF et al (2005) Increased levels of erucic acid in Brassica carinata by co-suppression and antisense repression of the endogenous FAD2 gene. Metabolic Eng 7:215–220CrossRefGoogle Scholar
  40. Jones L, Hamilton AJ, Voinnet O et al (1999) RNA-DNA interactions and DNA methylation in post-transcriptional gene silencing. Plant Cell 11:2291–2301CrossRefPubMedGoogle Scholar
  41. Kasschau KD, Fahlgren N, Chapman EJ et al (2007) Genome-wide profiling and analysis of Arabidopsis siRNAs. PLoS Biol 5:e57CrossRefPubMedGoogle Scholar
  42. Koornneef M (2002) Classical mutagenesis in higher plants. In: Gilmartin PM, Bowler C (eds) Molecular plant biology, vol 1, pp 1–11. Oxford, GB, Oxford University PressGoogle Scholar
  43. Koornneef M, Dellaert LMW, van der Veen JH (1982) EMS-induced and radiation-induced mutation frequencies at individual loci in Arabidopsis-thaliana (L) Heynh. Mutat Res 93:109–123PubMedGoogle Scholar
  44. Liljegren SJ, Roeder AH, Kempin SA et al (2004) Control of fruit patterning in Arabidopsis by INDEHISCENT. Cell 116:843–853CrossRefPubMedGoogle Scholar
  45. Lin X, Ruan X, Anderson MG et al (2005) siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res 33:4527–4535CrossRefPubMedGoogle Scholar
  46. Liu Y, Schiff M, Dinesh-Kumar SP (2002a) Virus-induced gene silencing in tomato. Plant J 31:777–786CrossRefPubMedGoogle Scholar
  47. Liu Y, Schiff M, Marathe R et al (2002b) Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 30:415–429CrossRefPubMedGoogle Scholar
  48. Liu Q, Singh SP, Green AG (2002) High-stearic and High-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing. Plant Physiol 129:1732–1743CrossRefPubMedGoogle Scholar
  49. Lu R, Martin-Hernandez AM, Peart JR et al (2003) Virus-induced gene silencing in plants. Methods 30:296–303CrossRefPubMedGoogle Scholar
  50. Lysak MA, Koch MA, Pecinka A et al (2005) Chromosome triplication found across the tribe Brassiceae. Genome Res 15:516–525CrossRefPubMedGoogle Scholar
  51. McCallum CM, Comai L, Greene EA et al (2000) Targeting induced local lesions IN genomes (TILLING) for plant functional genomics. Plant Physiol 123:439–442CrossRefPubMedGoogle Scholar
  52. Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431:343–349CrossRefPubMedGoogle Scholar
  53. Mietkiewska E, Hoffman TL, Brost JM et al (2008) Hairpin-RNA mediated silencing of endogenous FAD2 gene combined with heterologous expression of crambe abyssinica FAE gene causes an increase in the level of erucic acid in transgenic Brassica carinata seeds. Mol Breed 22:619–627CrossRefGoogle Scholar
  54. Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone syntahse gene into petunia results in revesible co-suppression of homologous genes in trans. Plant Cell 2:279–289CrossRefPubMedGoogle Scholar
  55. Niu QW, Lin SS, Reyes JL et al (2006) Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol 24:1420–1428CrossRefPubMedGoogle Scholar
  56. Oleykowski CA, Bronson Mullins CR, Godwin AK et al (1998) Mutation detection using a novel plant endonuclease. Nucleic Acids Res 26:4597–4602CrossRefPubMedGoogle Scholar
  57. Ossowski S, Schwab R, Weigel D (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J 53:674–690CrossRefPubMedGoogle Scholar
  58. Østergaard L, Kempin SA, Bies D et al (2006) Pod shatter-resistant Brassica fruit produced by ectopic expression of the FRUITFULL gene. Plant Biotechnol J 4:45–51CrossRefPubMedGoogle Scholar
  59. Østergaard L, King GJ (2008) Standardized gene nomenclature for the Brassica genus. Plant Methods 4:10CrossRefPubMedGoogle Scholar
  60. Penmetsa RV, Cook DR (2000) Production and characterization of diverse developmental mutants of Medicago truncatula. Plant Physiol 123:1387–1398CrossRefPubMedGoogle Scholar
  61. Perry JA, Wang TL, Welham TJ et al (2003) A TILLING reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus. Plant Physiol 131:866–871CrossRefPubMedGoogle Scholar
  62. Petersen BO, Albrechtsen M (2005) Evidence implying only unprimed RdRP activity during transitive gene silencing in plants. Plant Mol Biol 58:575–583CrossRefPubMedGoogle Scholar
  63. Pflieger S, Blanchet S, Camborde L et al (2008) Efficient virus-induced gene silencing in Arabidopsis using a “one-step” TYMV-derived vector. Plant J 56:678–690CrossRefPubMedGoogle Scholar
  64. Piccin A, Salameh A, Benna C et al (2001) Efficient and heritable functional knock-out of an adult phenotype in Drosophila using a GAL4-driven hairpin RNA incorporating a heterologous spacer. Nucleic Acids Res 29:E55–55CrossRefPubMedGoogle Scholar
  65. Qi Y, Denli AM, Hannon GJ (2005) Biochemical specialization within Arabidopsis RNA silencing pathways. Mol Cell 19:421–428CrossRefPubMedGoogle Scholar
  66. Qi Y, He X, Wang XJ, Kohany O, Jurka J, Hannon GJ (2006) Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443:1008–1012CrossRefPubMedGoogle Scholar
  67. Ratcliff FG, MacFarlane SA, Baulcombe DC (1999) Gene silencing without DNA. RNA-mediated cross-protection between viruses. Plant Cell 11:1207–1216CrossRefPubMedGoogle Scholar
  68. Schwab R, Ossowski S, Riester M et al (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133CrossRefPubMedGoogle Scholar
  69. Schwab R, Palatnik JF, Riester M et al (2005) Specific effects of MicroRNAs on the plant transcriptome. Dev Cell 8:517–527CrossRefPubMedGoogle Scholar
  70. Shivaprasad PV, Rajeswaran R, Blevins T, Schoelz J, Meins F Jr, Hohn T, Pooggin MM (2008) The CaMV transactivator/viroplasmin interferes with RDR6-dependent trans-acting and secondary siRNA pathways in Arabidopsis. Nucleic Acids Res 36:5896–5909CrossRefPubMedGoogle Scholar
  71. Slade AJ, Fuerstenberg SI, Loeffler D et al (2005) A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat Biotechnol 23:75–81CrossRefPubMedGoogle Scholar
  72. Slotkin RK, Martienssen R (2007) Transposable elements and the epigenetic regulation of the genome. Nat Rev Genet 8:272–285CrossRefPubMedGoogle Scholar
  73. Small I (2007) RNAi for revealing and engineering plant gene functions. Curr Opin Biotechnol 18:148–153CrossRefPubMedGoogle Scholar
  74. Stoutjesdijk PA, Singh SP, Liu Q et al (2002) hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing. Plant Physiol 129:1723–1731CrossRefPubMedGoogle Scholar
  75. Sundaresan V, Springer P, Volpe T et al (1995) Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements. Genes Dev 15:1797–1810CrossRefGoogle Scholar
  76. Till BJ, Cooper J, Tai TH et al (2007) Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol 7:19CrossRefPubMedGoogle Scholar
  77. Till BJ, Reynolds SH, Greene EA et al (2003) Large-scale discovery of induced point mutations with high-throughput TILLING. Genome Res 13:524–530CrossRefPubMedGoogle Scholar
  78. Till BJ, Reynolds SH, Weil C et al (2004) Discovery of induced point mutations in maize genes by TILLING. BMC Plant Biol 4:12CrossRefPubMedGoogle Scholar
  79. Turnage MA, Muangsan N, Peele CG et al (2002) Geminivirus-based vectors for gene silencing in Arabidopsis. Plant J 30:107–114CrossRefPubMedGoogle Scholar
  80. van der Krol AR, Lenting PE, Veenstra J et al (1988) An antisense chalcone synthase gene in transgenic plants inhibits flower pigmentation. Nature 333:866–869CrossRefGoogle Scholar
  81. van der Krol AR, Mur LA, Beld M et al (1990) Flavonoid genes in petunia: addition of a limited number of gene copies may lead to suppression of gene expression. Plant Cell 2:291–299CrossRefPubMedGoogle Scholar
  82. Vaucheret H (2006) Post-transcriptional small RNA pathways in plants: mechanisms and regulations. Genes Dev 20:759–771CrossRefPubMedGoogle Scholar
  83. Voinnet O (2005) Induction and suppression of RNA silencing: insights from viral infections. Nat Rev Genet 6:206–220CrossRefPubMedGoogle Scholar
  84. Wang N, Wang Y, Tian F et al (2008) A functional genomics resource for Brassica napus: development of an EMS mutagenized population and discovery of FAE1 point mutations by TILLING. New Phytol 180:751–765CrossRefPubMedGoogle Scholar
  85. Waterhouse PM, Wang MB, Lough T (2001) Gene silencing as an adaptive defence against viruses. Nature 411:834–842CrossRefPubMedGoogle Scholar
  86. Watson JM, Fusaro AF, Wang M et al (2005) RNA silencing platforms in plants. FEBS Lett 579:5982–5987CrossRefPubMedGoogle Scholar
  87. Wesley SV, Helliwell CA, Smith NA et al (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581–590CrossRefPubMedGoogle Scholar
  88. Wielopolska A, Townley H, Moore I et al (2005) A high-throughput inducible RNAi vector for plants. Plant Biotechnol J 3:583–590CrossRefPubMedGoogle Scholar
  89. Winkler S, Schwabedissen A, Backasch D et al (2005) Target-selected mutant screen by TILLING in Drosophila. Genome Res 15:718–723CrossRefPubMedGoogle Scholar
  90. Xie Z, Johansen LK, Gustafson AM et al (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2:E104CrossRefPubMedGoogle Scholar
  91. Xu P, Zhang Y, Kang L et al (2006) Computational estimation and experimental verification of off-target silencing during posttranscriptional gene silencing in plants. Plant Physiol 142:429–440CrossRefPubMedGoogle Scholar
  92. Yu B, Lydiate DJ, Young LW et al (2008) Enhancing the carotenoid content of Brassica napus seeds by downregulating lycopene epsilon cyclase. Transgenic Res 17:573–585CrossRefPubMedGoogle Scholar
  93. Zhang C, Ghabrial SA (2006) Development of Bean pod mottle virus-based vectors for stable protein expression and sequence-specific virus-induced gene silencing in soybean. Virology 344:401–411CrossRefPubMedGoogle Scholar
  94. Zhang C, Yang C, Whitham SA et al (2009) Development and Use of an Efficient DNA-Based Viral Gene Silencing Vector for Soybean. Mol Plant Microbe Interact 22:123–131CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Thomas Wood
    • 1
    Email author
  • Pauline Stephenson
    • 1
  • Lars Østergaard
    • 1
  1. 1.John Innes CentreNorwichUK

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