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The Sequencing-Based Mapping Method for Effectively Cloning Plant Mutated Genes.

ABSTRACT: A forward genetic approach is a powerful tool for identifying the genes underlying the phenotypes of interest. However, the conventional map-based cloning method is lengthy, requires a large mapping population and confirmation of many candidate genes in a broad genetic region to clone the causal variant. The whole-genome sequencing method clones the variants with a certain failure probability for multiple reasons, especially for heterozygotes, and could not be used to clone the mutation of epigenetic modifications. Here, we applied the highly complementary characteristics of these two methods and developed a sequencing-based mapping method (SBM) for identifying the location of plant variants effectively with a small population and low cost, which is very user-friendly for most popular laboratories. This method used the whole-genome sequencing data of two pooled populations to screen out enough markers. These markers were used to identify and narrow the candidate region by analyzing the marker-indexes and recombinants. Finally, the possible mutational sites were identified using the whole-genome sequencing data and verified in individual mutants. To elaborate the new method, we displayed the cloned processes in one Arabidopsis heterozygous mutant and two rice homozygous mutants. Thus, the sequencing-based mapping method could clone effectively different types of plant mutations and was a powerful tool for studying the functions of plant genes in the species with known genomic sequences.

SUBMITTER: Yu L

PROVIDER: S-EPMC8226582 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome-wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material.We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species.As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice.Plant-RRBS offers high-throughput and broad, genome-dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations.

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The Sequencing-Based Mapping Method for Effectively Cloning Plant Mutated Genes.

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