Application of a simplified method of chloroplast enrichment to small amounts of tissue for chloroplast genome sequencing

Applications in Plant Sciences
Shota SakaguchiYuji Isagi

Abstract

High-throughput sequencing of genomic DNA can recover complete chloroplast genome sequences, but the sequence data are usually dominated by sequences from nuclear/mitochondrial genomes. To overcome this deficiency, a simple enrichment method for chloroplast DNA from small amounts of plant tissue was tested for eight plant species including a gymnosperm and various angiosperms. Chloroplasts were enriched using a high-salt isolation buffer without any step gradient procedures, and enriched chloroplast DNA was sequenced by multiplexed high-throughput sequencing. Using this simple method, significant enrichment of chloroplast DNA-derived reads was attained, allowing deep sequencing of chloroplast genomes. As an example, the chloroplast genome of the conifer Callitris sulcata was assembled, from which polymorphic microsatellite loci were isolated successfully. This chloroplast enrichment method from small amounts of plant tissue will be particularly useful for studies that use sequencers with relatively small throughput and that cannot use large amounts of tissue (e.g., for endangered species).

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Citations

Nov 28, 2018·Scientific Reports·Carolina Osuna-MascaróFrancisco Perfectti
Jul 29, 2020·Frontiers in Plant Science·Michelle Zavala-PáezEric de Camargo Smidt
Dec 29, 2020·Mitochondrial DNA. Part B, Resources·Huan LiXueyi Zhu
Aug 25, 2021·PloS One·Raquel Santos da SilvaLeila do Nascimento Vieira

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Datasets Mentioned

BETA
KYO
00019999
727065
DRA005207
DRA005214
AP017904
AP017911

Methods Mentioned

BETA
chips
PCR
electrophoresis

Software Mentioned

BLASTN
Primer3
PipMaker
GenomeVx
CPGAVAS AnnoGenome
MSATCOMMANDER
MITObim
CLC Genomics Workbench

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