Chloroplast DNA (cpDNA) Sequencing

1. Why Conduct Chloroplast DNA Sequencing?

Chloroplast DNA sequencing plays a pivotal role in various realms such as plant taxonomy, phylogenetic analyses, population genetics, biodiversity conservation, and agronomic improvement. This methodology empowers us to illuminate the intricate evolutionary connections within plant species and explore genetic diversity, facilitating the identification and enhancement of superior plant varieties.

2. How Does Chloroplast DNA Differ from Nuclear DNA?

Chloroplast DNA, a compact circular molecule ensconced within the chloroplast, chiefly orchestrates vital processes like photosynthesis. In contrast, nuclear DNA abides within the cell nucleus, serving as the primary genetic reservoir in plants housing the majority of genes and genetic data.

3. How to Extract Chloroplast DNA?

The extraction of chloroplast DNA entails the isolation of chloroplasts from plant tissues, succeeded by the extraction of DNA from these organelles. Techniques commonly employed include mechanical disruption of plant leaves, differential centrifugation for chloroplast isolation, and DNA purification through methods such as CTAB or dedicated commercial kits.

4. What are the Common Chloroplast DNA Sequencing Methods?

Common sequencing methods include traditional Sanger sequencing and next-generation sequencing (NGS) technologies like Illumina, PacBio, and Oxford Nanopore. NGS technologies have emerged as the mainstream choice for chloroplast DNA sequencing due to their high throughput and relatively lower costs.

5. How to conduct quality control and contamination assessment?

Quality control steps include using software such as FastQC to check the read length and quality of sequencing data, and removing low-quality reads. Contamination assessment involves aligning reads to known databases to detect possible exogenous DNA contamination.

6. What is personalized analysis?

Personalized analysis is a customized analysis of sequencing data based on specific research needs. For example, in-depth study can be conducted on specific genes, or differences in chloroplast genomes of different samples can be compared.

7. What is Refseq chloroplast database alignment?

Refseq chloroplast database alignment is aligning sequencing data to known chloroplast genome sequences in the Refseq database for gene annotation, variant detection, and phylogenetic analysis. This helps to confirm the accuracy and completeness of sequencing results.

8. What are the applications of chloroplast DNA sequencing?

• Phylogenetics: Unraveling the evolutionary relationships among plants.
• Population Genetics: Investigating genetic diversity within populations and gene flow.
• Conservation Biology: Formulating conservation strategies to safeguard endangered species.
• Agriculture and Breeding: Selecting and improving superior crop varieties for agricultural advancement.

These applications underscore the versatility and significance of chloroplast DNA sequencing in elucidating evolutionary, ecological, and agricultural phenomena.

Chloroplast genome assemblies and comparative analyses of commercially important Vaccinium berry crops

Journal: Scientific Reports

Impact factor: 4.6

Published: 14 December 2022

Background

The genus Vaccinium includes over 450 species with major commercial crops such as blueberries, cranberries, bilberries, and lingonberries. These berries are economically important and widely consumed for their nutritional value. Taxonomic classification within Vaccinium is complex due to phenotypic variability and hybridization. Chloroplast genome sequencing offers potential for better taxonomic resolution, breeding, biotechnology, and product authentication. This study reports new chloroplast genomes for key Vaccinium species, providing valuable resources for future research and practical applications.

Methods

Results

Chloroplast genomes for five Vaccinium species were assembled using PacBio long reads for SHB and rabbiteye, resulting in complete gapless sequences. For NHB, lowbush, and bilberry, Illumina short reads were used, yielding assemblies with some gaps. Final cpDNA sequences exhibited a quadripartite structure, including large and small single copy regions, and a pair of inverted repeats.

Fig 1. Circular chloroplast genome map of southern highbush blueberry cv. 'Arcadia' (V. corymbosum hybrids).

Fig 2. Multiple sequence alignment of Vaccinium chloroplast genomes performed with mVISTA.

The chloroplast DNA (cpDNA) of five Vaccinium species (SHB, NHB, rabbiteye, lowbush, and bilberry) was annotated alongside the cranberry cpDNA from GenBank. Each cpDNA contained 112 unique functional genes, with some variability in gene copy numbers. Most genes were found in the large single copy (LSC) region, while the small single copy (SSC) and inverted repeats (IRs) contained fewer genes.

Comparative analysis showed high conservation and synteny among the cpDNAs, with most differences in non-coding regions and around IR borders. Simple sequence repeats (SSRs) and insertion-deletion polymorphisms were analyzed, identifying 10 polymorphic SSR loci useful for species differentiation. Two specific markers, "rpl36-rps8" and "rpoB-rpoA (3)," were identified to discriminate all six commercially relevant Vaccinium species.

Conclusion

This study assembled chloroplast genomes of five key Vaccinium species, revealing high conservation but some variability in non-coding regions. Comparative analyses identified regions useful for distinguishing these species and highlighted evolutionary patterns within highbush blueberries. More chloroplast genomes will aid further study and understanding of Vaccinium crop evolution.

Reference:

1. Fahrenkrog, A.M., Matsumoto, G.O., Toth, K. et al. Chloroplast genome assemblies and comparative analyses of commercially important Vaccinium berry crops. Sci Rep 12, 21600 (2022).