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.
Highlights
Doubled 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
Development of polymorphic markers for loss of heterozygosity screens To evaluate the practical use of an enzymatic mismatch cleavage approach for the screening of homozygosity in putative doubled haploid (DH) lines of barley, we carried out a pilot experiment to determine the presence of nucleotide polymorphisms in parental plants
We tested the parental lines Golden Promise and HOR1606 and synthetic mixtures of the two parental genomic DNAs with a total of 26 primer pairs that were previously developed for different barley TILLING projects (Table 1)
Summary
Doubled 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. The approach is especially powerful in crop breeding because haploid plants are in many cases made diploid either spontaneously or via treatment with chemicals such as colchicine. This allows the generation and rapid fixation of genetic variants in a homozygous state. Doubled haploidy could be induced in Arabidopsis thaliana by modification of the centromere-specific histone CENH3 [15] In theory, this approach could be applied to other species allowing great improvements in plant breeding
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