Regional mutagenesis using Dissociation in maize.

We describe genetic screens, molecular methods and web resources newly available to utilize Dissociation (Ds) as an insertional mutagen in maize. Over 1700 Ds elements have been distributed throughout the maize genome to serve as donor elements for local or regional mutagenesis. Two genetic screens are described to identify Ds insertions in genes-of-interest (goi). In scheme I, Ds is used to generate insertion alleles when a recessive reference allele is available. A Ds insertion will enable the cloning of the target gene and can be used to create an allelic series. In scheme II, Ds insertions in a goi are identified using a PCR-based screen to identify the rare insertion alleles among a population of testcross progeny. We detail an inverse PCR protocol to rapidly amplify sequences flanking Ds insertion alleles and describe a high-throughput 96-well plate-based DNA extraction method for the recovery of high-quality genomic DNA from seedling tissues. We also describe several web-based tools for browsing, searching and accessing the genetic materials described. The development of these Ds insertion lines promises to greatly accelerate functional genomics studies in maize.

[1]  K. Meksem,et al.  The Handbook of Plant Functional Genomics , 2008 .

[2]  V. Walbot,et al.  A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2 , 1995, Nature.

[3]  V. Walbot,et al.  The Maize Handbook , 1994, Springer Lab Manuals.

[4]  M. Heinlein.  Excision patterns of Activator (Ac) and Dissociation (Ds) elements in Zea mays L.: implications for the regulation of transposition. , 1996, Genetics.

[5]  B. Mcclintock,et al.  Chromosome organization and genic expression. , 1951, Cold Spring Harbor symposia on quantitative biology.

[6]  E. Grotewold,et al.  Insertional mutagenesis of the maize P gene by intragenic transposition of Ac. , 1992, Genetics.

[7]  H. Dooner,et al.  Transposition Pattern of the Maize Element Ac from the Bz-M2(ac) Allele. , 1989, Genetics.

[8]  T. Brutnell,et al.  Subfunctionalization of PhyB1 and PhyB2 in the control of seedling and mature plant traits in maize. , 2007, The Plant journal : for cell and molecular biology.

[9]  T. Brutnell,et al.  Generating Novel Allelic Variation Through Activator Insertional Mutagenesis in Maize , 2007, Genetics.

[10]  C. Weil,et al.  Adjacent sequences influence DNA repair accompanying transposon excision in maize. , 1996, Genetics.

[11]  I. M. Greenblatt,et al.  A chromosome replication pattern deduced from pericarp phenotypes resulting from movements of the transposable element, modulator, in maize. , 1984, Genetics.

[12]  T. Brutnell,et al.  Ac-Immobilized, a Stable Source of Activator Transposase That Mediates Sporophytic and Gametophytic Excision of Dissociation Elements in Maize , 2005, Genetics.

[13]  Somatic variegation and germinal mutability reflect the position of transposable element Dissociation within the maize R gene. , 1993, Genetics.

[14]  R. Appels,et al.  Chromosome structure and function — impact of new concepts , 1989 .

[15]  Xianghe Yan,et al.  Use of the Transposon Ac as a Gene-Searching Engine in the Maize Genome , 2002, The Plant Cell Online.

[16]  C. Weil,et al.  Novel, developmentally specific control of Ds transposition in maize , 1997, Molecular and General Genetics MGG.

[17]  W. Sheridan,et al.  Maize stocks modified to enhance the recovery of Ac-induced mutations , 1999 .

[18]  A. Klein,et al.  Excision and transposition of two Ds transposons from the bronze mutable 4 Derivative 6856 allele of Zea mays L. , 1990, Molecular and General Genetics MGG.

[19]  T. Brutnell,et al.  Activator Mutagenesis of the Pink scutellum1/viviparous7 Locus of Maize Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010249. , 2003, The Plant Cell Online.

[20]  S. Wessler,et al.  Molecular consequences of Ds insertion into and excision from the helix-loop-helix domain of the maize R gene. , 1998, Genetics.

[21]  S. Dellaporta,et al.  Reconstitutional mutagenesis of the maize P gene by short-range Ac transpositions. , 1992, Genetics.