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Study of the U6 small nuclear RNA genes in the Citrus limon (L.) Osbeck genome and amplification of the U6 gene regions among Citrus spp.


Citation :- Study of the U6 small nuclear RNA genes in the Citrus limon (L.) Osbeck genome and amplification of the U6 gene regions among Citrus spp.. Res. Crop. 26: 604-610
OLEG S. ALEXANDROV AND DMITRY V. ROMANOV olegsandrov@gmail.com
Address : All-Russia Research Institute of Agricultural Biotechnology, 127550 Moscow, Russia
Submitted Date : 27-09-2025
Accepted Date : 16-10-2025

Abstract

The small nuclear RNA gene U6 is required for spliceosome formation in cells. Being "housekeeping genes," these genes are relatively conserved across species, but have highly variable promoter regions. In practice, the promoters of U6 genes are used in vectors for genome editing, ensuring the synthesis of guide RNA in the cell of the organism being edited. In many CRISPR/Cas9 vectors, the arabidopsis U6 gene promoter is used, but replacing it with endogenous promoters often increases the efficiency of genome editing. Citrus plants are important crops worldwide, so improving genome editing systems for them is highly relevant. Therefore, the aim of this work was to search for lemon-specific endogenous promoters of U6 genes and their amplification in different citrus plant genomes. BLAST analysis with the tomato U6 gene sequence revealed six loci in the lemon genome that contained the U6 gene. The sequences of these loci became the basis for the design of specific primers. Primers were tested using electronic and classical PCR. In most Citrus species, experiments showed single-copy nature of the studied loci and correspondence between the lengths of expected and obtained PCR products. Only in the case of the ‘Buddha's Hand’ citron variety, there was no amplification with primers Le62_1f/Le62_1r and there was amplification of two fragments with primers Le63_1f/Le63_1r, the lengths of which did not correspond to the expected ones. Overall, the primers developed and presented in this article may be useful to improve the vectors for genome editing of citrus plants, as well as to perform fundamental studies of U6 genes.

Keywords

Citrus plants genome editing lemon promoters single-copy loci U6 genes

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