Validation of doubled haploid plants by enzymatic mismatch cleavage

Abstract

BackgroundDoubled haploidy is a fundamental tool in plant breeding as it provides the fastest way to generate populations of meiotic recombinants in a genetically fixed state. A wide range of methods has been developed to produce doubled haploid (DH) plants and recent advances promise efficient DH production in otherwise recalcitrant species. Since the cellular origin of the plants produced is not always certain, rapid screening techniques are needed to validate that the produced individuals are indeed homozygous and genetically distinct from each other. Ideal methods are easily implemented across species and in crops where whole genome sequence and marker resources are limited.ResultsWe have adapted enzymatic mismatch cleavage techniques commonly used for TILLING (Targeting Induced Local Lesions IN Genomes) for the evaluation of heterozygosity in parental, F1 and putative DH plants. We used barley as a model crop and tested 26 amplicons previously developed for TILLING. Experiments were performed using self-extracted single-strand-specific nuclease and standard native agarose gels. Eleven of the twenty-six tested primers allowed unambiguous assignment of heterozygosity in material from F1 crosses and loss of heterozygosity in the DH plants. Through parallel testing of previously developed Simple Sequence Repeat (SSR) markers, we show that 3/32 SSR markers were suitable for screening. This suggests that enzymatic mismatch cleavage approaches can be more efficient than SSR based screening, even in species with well-developed markers.ConclusionsEnzymatic mismatch cleavage has been applied for mutation discovery in many plant species, including those with little or no available genomic DNA sequence information. Here, we show that the same methods provide an efficient system to screen for the production of DH material without the need of specialized equipment. This gene target based approach further allows discovery of novel nucleotide polymorphisms in candidate genes in the parental lines.

A Protocol for Validation of Doubled Haploid Plants by Enzymatic Mismatch Cleavage

“…Mixtures of genomic DNA from a DH plant and GP show cleavage products, while mixture of the same material with HOR1606 does not, indicating the DH harbours the HOR1606 allele (lanes 14 and 15). This figure and legend are reproduced from (Hofinger et al 2013) similar results, use the cheaper version. Some Taq polymerases, like TaKaRa Ex Taq, come supplied with dNTPs and buffer containing MgCl 2 .…”

“…As a proof of principle, Hofinger et al (2013) developed this method for barley and compared it to another approach for validation of DH plants: simple sequence repeat (SSR) markers (Hofinger et al 2013). In this work, the authors showed that 11/26 primer pairs were suitable for DH validation by enzymatic mismatch cleavage, while only 3/32 previously characterized SSR primer sets were suitable.…”

“…Indeed, in addition to barley, we show data for screening putative DH Eragrostis tef plants (Fig. 16.3 and Hofinger et al 2013). While the methods described in this protocol are straightforward and low cost, a key component of successful application is proper experimental design.…”

Example Data

“…8.6 Mismatch cleavage with crude enzyme extracts containing single-strand-specific nucleases prepared from weedy plants (W) or celery (C). PCR products of the target gene nb2-rdg2a (1,500-bp-PCR product) were produced from genomic DNA of barley containing a known SNP (Hofinger et al 2013). The PCR products were digested with weed and celery enzyme extracts at different concentrations (listed above sample).…”

“…8.4 PCR amplification of genomic DNAs described in Chap. 7 using primers for the barley nb2-rdg2a (top panel) and nbs3-rdg2a (bottom panel) gene targets as described in Hofinger et al (2013). Samples are loaded in the same order as in Fig.…”