ASAP: amplification, sequencing & annotation of plastomes

Area of Science:

• Plant genomics
• Molecular biology
• Bioinformatics

Background:

• Plastid genome sequencing is crucial for genomics studies but traditionally involves complex DNA isolation and cloning procedures.
• Current methods like rolling circle amplification (RCA) require purified chloroplast DNA and subsequent shearing and cloning.

Purpose of the Study:

• To develop a universal, high-throughput, rapid, and cost-effective PCR-based technique for amplifying, sequencing, and assembling plastid genomes from total plant DNA.
• To demonstrate the method's utility for structural and comparative genomics across diverse plant species.

Main Methods:

• Utilized conserved coding or intergenic regions of plastid genes to design primer pairs.
• Identified conserved regions through bioinformatic alignment of available eudicot plastomes.
• Generated overlapping amplicons (1-1.2 kb) from the inverted repeat region of 14 diverse genera.

Main Results:

• Achieved 100% coverage of the inverted repeat region in several species including Arabidopsis, tobacco, and tomato.
• Obtained over 80% coverage in more distant species like Ginkgo and Equisetum.
• Identified large deletions in tomato and Amaranthus plastomes relative to tobacco, highlighting the method's utility for comparative genomics.

Conclusions:

• The developed method provides simple, inexpensive, and rapid access to plastid sequence data.
• It serves as a universal platform for plastid genome characterization and increases experimental throughput.
• The technique is valuable for whole genome studies and assessing interspecies variability in plastid genomics.