A New Chloroplast DNA Extraction Protocol Significantly Improves the Chloroplast Genome Sequence Quality of Foxtail Millet (Setaria italica (L.) P. Beauv.)

Abstract

The complexity of the leaf constitution of foxtail millet (Setaria italica (L.) P. Beauv.) makes it difficult to obtain high-purity cpDNA. Here, we developed a protocol to isolate high-quality cpDNA from foxtail millet and other crops. The new protocol replaces previous tissue grinding and homogenization by enzyme digestion of tiny leaf strips to separate protoplasts from leaf tissue and protects chloroplasts from damage by undue grinding and homogenization and from contamination of cell debris and nuclear DNA. Using the new protocol, we successfully isolated high-quality cpDNAs for whole-genome sequencing from four foxtail millet cultivars, and comparative analysis revealed that they were approximately 27‰ longer than their reference genome. In addition, six cpDNAs of four other species with narrow and thin leaf blades, including wheat (Triticum aestivum L.), maize (Zea may L.), rice (Oryza sativa L.) and sorghum (Sorghum bicolor (L.) Moench), were also isolated by our new protocol, and they all exhibited high sequence identities to their corresponding reference genomes. A maximum-likelihood tree based on the chloroplast genomes we sequenced here was constructed, and the result was in agreement with previous reports, confirming that these cpDNA sequences were available for well-supported phylogenetic analysis and could provide valuable resources for future research.

Phylogenomic Analysis of the Plastid Genome of the Peruvian Purple Maize Zea mays subsp. mays cv. ‘INIA 601’

“…Some maize plastid genomes are publicly available, including one partial ancient genome (SM10) [ 19 ], four Zea species, the subspecie Zea mays subsp. huehuetenangensis (Iltis & Doebley) Doebley, 1990 [ 20 ], and several domesticated maize lines including ‘B73’ [ 16 , 17 , 18 , 19 , 20 , 21 ], ‘B37’ (C, T, S, N), ‘A188’ [ 22 ], and ‘Zhengdan958’ [ 23 ]. Additionally, the plastid genomes of the close relatives Tripsacum dactyloides (L.) L [ 24 ] and Sorgum bicolor (L.) Moench [ 17 ] are also available.…”

Chloroplast genome, nuclear ITS regions, mitogenome regions, and Skmer analysis resolved the genetic relationship among Cinnamomum species in Sri Lanka

“…Regular DNA extraction protocols such as CTAB [ 76 ] and SDS [ 77 ] result in total cellular DNA, including genomic, chloroplast, and mitochondrial DNA. However, there are protocols available for additional enrichment of chloroplast DNA, including plastid isolation, enrichment via methylation-sensitive capture, hybrid bait capture, and PCR [ 78 , 79 ]. Nevertheless, these isolation or enrichment procedures are time-consuming and expensive [ 80 ].…”

Phylogenomic relationships and species identification of the olive genus Olea (Oleaceae)

“…Owing to the development of sequencing methods and the decreasing costs of genome sequencing, the barcode concept can be extended from one or more genes to the entire genome (Coissac et al, 2016). The chloroplast genome is easily obtained using PCR enrichment (Dong et al, 2013), direct isolation (Shi et al, 2012; Liu et al, 2019), or skimming of genomic DNA (Zeng et al, 2018) compared with nuclear and mitochondrial genomes. The chloroplast genome contained as much variation as the COI locus in animals and many studies have argued for it as the plant super‐barcode (Hollingsworth et al, 2016; Fu et al, 2019; Ji et al, 2019; Shen et al, 2019).…”