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PubMed2025

Whole-Genome Bisulfite Sequencing with a Small Amount of DNA.

Whole-genome bisulfite sequencing (WGBS) is the most widely used method to study DNA methylation profiles across the genome. Since the bisulfite reaction causes DNA degradation, a new approach called post-bisulfite adapter tagging (PBAT) was developed to overcome this problem by adding adapters after bisulfite treatment. In mammals, the PBAT method is used for single-cell bisulfite sequencing (scBS-seq), which enables DNA methylation analysis using a very small amount of DNA from only a few cells, including single-cell input. This protocol involves bisulfite conversion, followed by preamplification and tagging with random hexamer primers prior to Illumina library preparation. Since many procedures are completed in one single test tube, the loss of DNA can be minimized, enabling highly sensitive experiments to study DNA methylation profiles from a very small amount of input material.

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PubMed2025

Functional genetics of rice PISTILLATA genes reveals new roles and target genes in flowering time, female fertility, and parthenocarpy.

Floral organ identity is controlled largely by the combinatorial action of MADS domain homeotic transcription factors. Lodicules are specialized plant organs in cereals and grasses that are involved in floret opening and facilitate pollination and fertility in rice (Oryza sativa L.). To understand the mechanisms underlying the specification of the rice lodicule, we investigated the developmental functions of the rice PISTILLATA (PI) paralogs, OsMADS2, and OsMADS4. Null osmads2 mutants reiterated OsMADS2 nonredundant lodicule specification roles and revealed new roles in flowering time and floral organ number and fate. Doubly perturbed osmads2 osmads4kd florets had severe abnormalities, were female infertile, yet could initiate parthenocarpy. Ubiquitous OsMADS4 overexpression rescued osmads2 abnormalities. We also utilized genome-wide binding analyses and transcriptome profiling to identify putative target genes contributing to OsMADS2 functions. In osmads2d8/d8 null mutant, we observed deregulated genes in a plethora of processes including lodicule and stamen development, floral organ number, and cell wall development. Some examples are cell division regulators (Cyclin D6, Cyclin-P4-1-like), an aquaporin (PIP1A), a peptide transporter, a vascular developmental regulator (HOX1), and a cell wall modulator (GH9B16). The deregulation of these genes may be associated with the disrupted cell division, tissue differentiation, and physiology of the malformed lodicules in osmads2 and osmads2 osmads4kd florets. Altogether, we reveal novel roles for the rice PI paralogs in flowering time, panicle exsertion, and embryo sac differentiation, identify gene targets for lodicule development, and provide mechanistic insights on the functional diversification of rice PI paralogs.

Oryza↗