Dataset Information

Characterization of in vitro haploid and doubled haploid Chrysanthemum morifolium plants via unfertilized ovule culture for phenotypical traits and DNA methylation pattern.

ABSTRACT: Chrysanthemum is one of important ornamental species in the world. Its highly heterozygous state complicates molecular analysis, so it is of interest to derive haploid forms. A total of 2579 non-fertilized chrysanthemum ovules pollinated by Argyranthemum frutescens were cultured in vitro to isolate haploid progeny. One single regenerant emerged from each of three of the 105 calli produced. Chromosome counts and microsatellite fingerprinting showed that only one of the regenerants was a true haploid. Nine doubled haploid derivatives were subsequently generated by colchicine treatment of 80 in vitro cultured haploid nodal segments. Morphological screening showed that the haploid plant was shorter than the doubled haploids, and developed smaller leaves, flowers, and stomata. An in vitro pollen germination test showed that few of the haploid's pollen were able to germinate and those which did so were abnormal. Both the haploid and the doubled haploids produced yellow flowers, whereas those of the maternal parental cultivar were mauve. Methylation-sensitive amplification polymorphism (MSAP) profiling was further used to detect alterations in cytosine methylation caused by the haploidization and/or the chromosome doubling processes. While 52.2% of the resulting amplified fragments were cytosine methylated in the maternal parent's genome, the corresponding proportions for the haploid's and doubled haploids' genomes were, respectively, 47.0 and 51.7%, demonstrating a reduction in global cytosine methylation caused by haploidization and a partial recovery following chromosome doubling.

SUBMITTER: Wang H

PROVIDER: S-EPMC4273617 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Characterization of in vitro haploid and doubled haploid Chrysanthemum morifolium plants via unfertilized ovule culture for phenotypical traits and DNA methylation pattern.

Wang Haibin H Dong Bin B Jiang Jiafu J Fang Weimin W Guan Zhiyong Z Liao Yuan Y Chen Sumei S Chen Fadi F

Frontiers in plant science 20141222

Chrysanthemum is one of important ornamental species in the world. Its highly heterozygous state complicates molecular analysis, so it is of interest to derive haploid forms. A total of 2579 non-fertilized chrysanthemum ovules pollinated by Argyranthemum frutescens were cultured in vitro to isolate haploid progeny. One single regenerant emerged from each of three of the 105 calli produced. Chromosome counts and microsatellite fingerprinting showed that only one of the regenerants was a true hapl ...[more]

PMID: 25566305

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Project description: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.

Project description:Customizable endonucleases are providing an effective tool for genome engineering. The resulting primary transgenic individuals (T0) are typically heterozygous and/or chimeric with respect to any mutations induced. To generate genetically fixed mutants, they are conventionally allowed to self-pollinate, a procedure which segregates individuals into mutant heterozygotes/homozygotes and wild types. The chances of recovering homozygous mutants among the progeny depend not only on meiotic segregation but also on the frequency of mutated germline cells in the chimeric mother plant. In Nicotiana species, the heritability of Cas9-induced mutations has not been demonstrated yet. RNA-guided Cas9 endonuclease-mediated mutagenesis was targeted to the green fluorescent protein (GFP) gene harbored by a transgenic tobacco line. Upon retransformation using a GFP-specific guide RNA/Cas9 construct, the T0 plants were allowed to either self-pollinate, or were propagated via regeneration from in vitro cultured embryogenic pollen which give rise to haploid/doubled haploid plants or from leaf explants that form plants vegetatively. Single or multiple mutations were detected in 80% of the T0 plants. About half of these mutations proved heritable via selfing. Regeneration from in vitro cultured embryogenic pollen allowed for homozygous mutants to be produced more efficiently than via sexual reproduction. Consequently, embryogenic pollen culture provides a convenient method to rapidly generate a variety of genetically fixed mutants following site-directed mutagenesis. The recovery of a mutation not found among sexually produced and analyzed progeny was shown to be achievable through vegetative plant propagation in vitro, which eventually resulted in heritability when the somatic clones were selfed. In addition, some in-frame mutations were associated with functional attenuation of the target gene rather than its full knock-out. The generation of mutants with compromised rather than abolished gene functionality holds promise for future approaches to the conclusive functional validation of genes which are indispensible for the plant.

Dataset Information

Characterization of in vitro haploid and doubled haploid Chrysanthemum morifolium plants via unfertilized ovule culture for phenotypical traits and DNA methylation pattern.

Publications

Characterization of in vitro haploid and doubled haploid Chrysanthemum morifolium plants via unfertilized ovule culture for phenotypical traits and DNA methylation pattern.

Similar Datasets

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