Creation of DNA libraries for high-throughput genotyping of Karelian birch and analysis of their quality

The article discusses the development of a practical laboratory protocol for creating DNA libraries for high-throughput genotyping of Karelian birch. Karelian birch wood (Betula pendula Roth var. carelica (Mercklin) Hämet-Ahti) has always been valued for its unique and distinctly patterned annual rings. This material is highly prized in manufacturing and is used in furniture, tableware, various interior items, and decorative inlays. Due to the complexity of identifying the type of wood at the early stages of tree growth, plantations with a large number of individuals have to be used. The process of selecting «Karelian» samples based on the external characteristics of the tree is challenging because it largely depends on the knowledge and experience of a breeding specialist. Making the selection of «Karelian» forms more precise and faster can be achieved with genetic markers that objectively reflect changes in the genome associated with the formation of patterned wood. Identifying the genetic loci that determine the «wood patterning» trait in seedlings at early stages of development would help in the future to create «pure» Karelian birch plantations without the presence of trees with regular wood. Genetic markers allow for a more precise selection of «Karelian» forms, excluding trees with regular wood. To develop such molecular markers, it is necessary to identify mutations in Karelian birch genes that distinguish it from pendulous birch and are linked to the inheritance of «patterned» wood. The technology of high-throughput genotyping based on sequencing (Genotyping By Sequencing, GBS) can be used to search for mutations. One of the GBS variants is the technology of reading short DNA sequences adjacent to specific restriction sites (RADseq, Restriction site-associated DNA sequencing). The development of a working laboratory protocol for creating RADseq libraries is a key step in high-throughput genotyping projects. The goal of this research is to describe the protocol for preparing a RADseq library using 12 Karelian birch DNA samples, as well as to analyze the quality of the obtained DNA fragment readings from the sequencing of the created library using the Illumina HiSeq2500 instrument. The results of the analysis showed that the protocol used allows for obtaining high-quality libraries from birch leaf DNA. This enables the progression to the next stages of analysis, including aligning reads to the reference sequence and searching for SNP markers.

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