identifying-mutations-high-resolution-melting-tilling-population

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the ...

Zhejiang University

CRISPR-S: an active interference element for a rapid and inexpensive selection of genome-edited, transgene-free rice plants.

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics strategy for the high-throughput screening of induced mutations. γ radiation, which often induces both insertion/deletion (Indel) and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting (HRM) analysis. Pooled rice (Oryza sativa) samples mixed at a 1:7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M plants is recommended for screening γ ray-induced mutants in rice. For demonstration, a γ ray-mutagenized M rice population (n=4560) was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution (G→A) and one single nucleotide insertion (A) were identified in OsLCT1, and one trinucleotide (TTC) deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ ray mutagenesis to the breeding of rice and other seed crops.

High-resolution melting-based TILLING of γ ray-induced mutations in rice.

N-methyladenosine (mA) messenger RNA methylation is a gene regulatory mechanism affecting cell differentiation and proliferation in development and cancer. To study the roles of mA mRNA methylation in cell proliferation and tumorigenicity, we investigated human endometrial cancer in which a hotspot R298P mutation is present in a key component of the methyltransferase complex (METTL14). We found that about 70% of endometrial tumours exhibit reductions in mA methylation that are probably due to either this METTL14 mutation or reduced expression of METTL3, another component of the methyltransferase complex. These changes lead to increased proliferation and tumorigenicity of endometrial cancer cells, likely through activation of the AKT pathway. Reductions in mA methylation lead to decreased expression of the negative AKT regulator PHLPP2 and increased expression of the positive AKT regulator mTORC2. Together, these results reveal reduced mA mRNA methylation as an oncogenic mechanism in endometrial cancer and identify mA methylation as a regulator of AKT signalling.

Song-Mei Liu

National Key Laboratory of Rice Biology,
Zhejiang Provincial Key Laboratory of Crop Genetic Resources,
Institute of Crop Science,
National Key Laboratory of Rice Biology, Zhejiang Provincial Key Laboratory of Crop Genetic Resources, Institute of Crop Science

Zhejiang University

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice