New Reverse Genetics Resources for Maize: Facile Production and Efficient Indexing using Next-Generation Sequencing Technology

A sequence-indexed reverse genetics resource is essential to fully exploit the maize genome sequence.  The objectives of our NSF-PGRP-funded project are to: (1) Sequence-index a collection of Ac and Ds transposants from bz-m2(D1) wx-m7(Ac); (2) Develop a set of 120 roughly equidistant transgenic Ds* launching platforms carrying markers that allow simple visual detection of element transposition from any region of the genome and, thus, enable researchers to generate regional gene knock-out collections; (3) Sequence-index several thousand Ds* insertion sites from model platforms using a method generalizable to any collection of insertions; and (4) Develop a web-searchable database of insertion site sequences cross-referenced to stocks available from the Maize Genetics Co-op.

So far, we have: (1) Mapped over 300 native tac (trAc) or tds (trDs) sites to the maize genome; (2) Using a C1 (colored seed) marker interrupted by a GFP-tagged Ds* element, 80 c1-m transgenic lines with Ds* transposition activity have been mapped to the reference B73 genome. (3) Over 10,000 trDs* have been selected from c1-m lines with high reversion frequencies.  A subset of the Ds-GFP flanking sites (dsg) has been mapped to B73 using next generation sequencing (NGS).  Of the first 1056 dsg sequences, 852 (81%) were unequivocally indexed to a specific cell in the 3-D pool and mapped, illustrating the efficiency of NGS and 3-D pooling for sequence-indexing a large collection of transposants.  (4) All the lines are listed in our web-searchable database (http://www.acdsinsertions.org/) and will be sent to the Maize Genetics Stock Center for distribution.