Chloroplasts are the most important and prevalent plastids in plant cells and are the key site for photosynthesis. Chloroplast genomic DNA is generally a double-stranded circular molecule, and very rarely linear. The structure of chloroplast genome is very conservative and generally contains four parts, short single copy (SSC), long single copy (LSC), inverted repeat IRA and IRB. Most of the IRA and IRB are 10-24 Kb long each and encode the same sequence in opposite directions. The chloroplast DNA (cpDNA) promoter is similar to that of prokaryotes, with some genes producing single cis-trans mRNAs and others multiple cis-trans mRNAs. During the long evolutionary process, the structural order of these parts has remained constant, and the differences between chloroplast genomes of different species are mainly in the length and orientation of the IR regions.
Traditionally, plant chloroplast genomes were obtained by designing simple primers using conserved sequences of chloroplast genomes, amplifying unknown sequences and long PCR amplification, sequencing the amplified products for one generation, and finally splicing and assembling to obtain complete chloroplast genomes. However, it is time-consuming. With the development of science and technology, sequencing means are constantly updated, and in recent years, many scientists are keen to use high-throughput sequencing, first isolate chloroplasts and then extract total DNA or cpDNA, select suitable chloroplast reference genome, design simple and parallel primers for PCR fishing, unknown sequence amplification and long PCR amplification. High-throughput sequencing is combined with one-generation Sanger sequencing to obtain the full sequence of chloroplast genome. In terms of years' professional experience in this field, Creative Biogene can provide you with the most affordable and high-quality chloroplast DNA sequencing services.
Figure 1. The process of chloroplast DNA sequencing.
Chloroplast genome structure and sequence information are of great value in revealing the origin of species, evolutionary and the affinities of different species.
Meanwhile, chloroplast transformation technology shows great potential in genetic improvement and production of biological agents, while chloroplast genome structure and sequence analysis are the cornerstones of chloroplast transformation.
High-throughput sequencing of plant chloroplast genomes breaks through the experimental barriers of traditional cpDNA isolation and purification, and can directly use total tissue DNA to obtain chloroplast genomes for in-depth analysis of sequence information, and obtain information on species classification, phylogeny, and geographic genealogy genetics through comparative genome analysis, including scanning map sequencing and completion map sequencing.