Deletion-based reverse genetics in Medicago truncatula

Abstract

The primary goal of reverse genetics, the identification of null mutations in targeted genes, is achieved through screening large populations of randomly mutagenized plants. T-DNA and transposon-based mutagenesis has been widely employed but is limited to species in which transformation and tissue culture are efficient. In other species, TILLING (for Targeting Induced Local Lesions IN Genomes), based on chemical mutagenesis, has provided an efficient method for the identification of single base pair mutations, only 5% of which will be null mutations. Furthermore, the efficiency of inducing point mutations, like insertion-based mutations, is dependent on target size. Here, we describe an alternative reverse genetic strategy based on physically induced genomic deletions that, independent of target size, exclusively recovers knockout mutants. Deletion TILLING (De-TILLING) employs fast neutron mutagenesis and a sensitive polymerase chain reaction-based detection. A population of 156,000 Medicago truncatula plants has been structured as 13 towers each representing 12,000 M2 plants. The De-TILLING strategy allows a single tower to be screened using just four polymerase chain reaction reactions. Dual screening and three-dimensional pooling allows efficient location of mutants from within the towers. With this method, we have demonstrated the detection of mutants from this population at a rate of 29% using five targets per gene. This De-TILLING reverse genetic strategy is independent of tissue culture and efficient plant transformation and therefore applicable to any plant species. De-TILLING mutants offer advantages for crop improvement as they possess relatively few background mutations and no exogenous DNA.

Figure 1.

A reconstruction experiment showing the amplification of the M. truncatula DMI1 locus from genomic DNA of wild type (A17), the dmi1-4 mutant (D1), and pools containing dmi1-4 and wild-type DNA at ratios of 1:25 ng (1:25), 1:1,000 ng (1K), 1:4,000 ng (4K), 1:8,000 ng (8K), 1:12,000 ng (12K), 1:16,000 ng (16K), 1:20,000 ng (20K), and 1:24,000 ng (24K). Nested PCR primers flank the 18-kb deletion by 350 bp (A), 3.0 kb (B), and 8.0 kb (C). Amplification from the wild-type region is suppressed entirely under these conditions, allowing amplification of the mutant product in pools of over 1:24,000 for the 350-bp and 3.0-kb assays and 1:12,000 genomes for the 8.0-kb assay. 1⁰, Primary PCR; 2⁰, secondary PCR.

Figure 2.

A reconstruction experiment showing the amplification of the M. truncatula NSP2 locus from genomic DNA of wild-type M. truncatula (A17), the nsp2-1 mutant (N2), and pools containing nsp2-1 and wild-type DNA at ratios of 1:25 ng (1:25), 1:1,000 ng (1K), 1:4,000 ng (4K), 1:8,000 ng (8K), 1:12,000 ng (12K), 1:16,000 ng (16K), 1:20,000 ng (20K), and 1:24,000 ng (24K). Only the secondary PCR products are shown in each case. A, Nested PCR. Wild-type alleles are preferentially amplified. B, Poison primer suppression enhances detection of the mutant allele in pools of up to 1:1,000 genomes. C, Restriction suppression. EcoRV-treated templates allow reliable detection in pools of up to 4,000 plants. D, De-TILLING strategy. Combining poison primer and restriction suppression allows preferential amplification of the mutant allele in pools containing a 24,000-fold excess of wild-type sequences. 1⁰, Primary PCR; 2⁰, secondary PCR.

Figure 3.

Structure of a De-TILLING tower. Each tower consists of 480 pools, each of which is genomic DNA of 25 seedlings taken from the pooled M2 progeny of five mutagenized M1 plants. Each tower is initially pooled into 25 row, column, and plate pools. These are used to create a pair of reciprocal HTPs. The De-TILLING population is initially screened using the 54 HTP representing the 13 towers twice. When a deletion mutant is detected, 25 3D pools are screened to locate the mutant to an individual well. The mutant is then recovered from the identified pool.

Figure 4.

Recovering a mutant for a LysM receptor-like kinase. A, Assays were designed around five restriction sites unique within 2- to 2.3-kb regions. The StyI assay (red) detected a 422-bp deletion within the population. B, Two identical PCR products indicate the presence of a deletion allele within tower 4. Note the spurious PCR products in towers 6 and 7 that are not real detection events. C, Amplification from the 3D pools of tower 4 locates a single M2 pool containing the mutant. D, PCR screening of 29 seedlings grown from this pools allow the lysM1-1 mutant to be recovered.

Figure 5.

Detection of the efd-1 mutant. A, Identical PCR products occurring in tower 4 reveal the presence of efd-1, an ERF transcription factor mutant possessing a 1,571-bp deletion. B, Amplification from the 3D pools reveals the row, column, and plate location of the efd-1 mutant containing M2 pool within tower 4.