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Searching for a Needle in a Haystack: Cas9-Targeted Nanopore Sequencing and DNA Methylation Profiling of Full-Length Glutenin Genes in a Big Cereal Genome

1
Laboratory of Marker-Assisted and Genomic Selection of Plants, All-Russia Research Institute of Agricultural Biotechnology, Timiryazevskaya Str. 42, 127550 Moscow, Russia
2
Kurchatov Genomics Center of ARRIAB, All-Russia Research Institute of Agricultural Biotechnology, Timiryazevskaya Str. 42, 127550 Moscow, Russia
3
N.V. Tsitsin Main Botanical Garden of the Russian Academy of Sciences, Botanicheskaya Str. 4, 127276 Moscow, Russia
*
Author to whom correspondence should be addressed.
Academic Editors: Yin Li, Guangxiao Yang, Yongfang Wan, Jian Zeng and Yaqiong Wang
Received: 25 November 2021 / Revised: 16 December 2021 / Accepted: 16 December 2021 / Published: 21 December 2021
(This article belongs to the Topic Plant Functional Genomics and Crop Genetic Improvement)
Sequencing and epigenetic profiling of target genes in plants are important tasks with various applications ranging from marker design for plant breeding to the study of gene expression regulation. This is particularly interesting for plants with big genome size for which whole-genome sequencing can be time-consuming and costly. In this study, we asked whether recently proposed Cas9-targeted nanopore sequencing (nCATS) is efficient for target gene sequencing for plant species with big genome size. We applied nCATS to sequence the full-length glutenin genes (Glu-1Ax, Glu-1Bx and Glu-1By) and their promoters in hexaploid triticale (X Triticosecale, AABBRR, genome size is 24 Gb). We showed that while the target gene enrichment per se was quite high for the three glutenin genes (up to 645×), the sequencing depth that was achieved from two MinION flowcells was relatively low (5–17×). However, this sequencing depth was sufficient for various tasks including detection of InDels and single-nucleotide variations (SNPs), read phasing and methylation profiling. Using nCATS, we uncovered SNP and InDel variation of full-length glutenin genes providing useful information for marker design and deciphering of variation of individual Glu-1By alleles. Moreover, we demonstrated that glutenin genes possess a ‘gene-body’ methylation epigenetic profile with hypermethylated CDS part and hypomethylated promoter region. The obtained information raised an interesting question on the role of gene-body methylation in glutenin gene expression regulation. Taken together, our work disclosures the potential of the nCATS approach for sequencing of target genes in plants with big genome size. View Full-Text
Keywords: nanopore sequencing; Cas9-enrichment; triticale; glutenin genes; DNA methylation nanopore sequencing; Cas9-enrichment; triticale; glutenin genes; DNA methylation
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MDPI and ACS Style

Kirov, I.; Polkhovskaya, E.; Dudnikov, M.; Merkulov, P.; Vlasova, A.; Karlov, G.; Soloviev, A. Searching for a Needle in a Haystack: Cas9-Targeted Nanopore Sequencing and DNA Methylation Profiling of Full-Length Glutenin Genes in a Big Cereal Genome. Plants 2022, 11, 5. https://doi.org/10.3390/plants11010005

AMA Style

Kirov I, Polkhovskaya E, Dudnikov M, Merkulov P, Vlasova A, Karlov G, Soloviev A. Searching for a Needle in a Haystack: Cas9-Targeted Nanopore Sequencing and DNA Methylation Profiling of Full-Length Glutenin Genes in a Big Cereal Genome. Plants. 2022; 11(1):5. https://doi.org/10.3390/plants11010005

Chicago/Turabian Style

Kirov, Ilya, Ekaterina Polkhovskaya, Maxim Dudnikov, Pavel Merkulov, Anastasia Vlasova, Gennady Karlov, and Alexander Soloviev. 2022. "Searching for a Needle in a Haystack: Cas9-Targeted Nanopore Sequencing and DNA Methylation Profiling of Full-Length Glutenin Genes in a Big Cereal Genome" Plants 11, no. 1: 5. https://doi.org/10.3390/plants11010005

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