Plant Gene and Trait 2025, Vol.16, No.1, 23-31 http://genbreedpublisher.com/index.php/pgt 27 5 Methodologies for Analyzing Chloroplast Markers 5.1 DNA extraction and amplification To analyze chloroplast markers, high-quality DNA should be extracted first. The modified CTAB method is very effective for Eucommia ulmoides and can extract genomic DNA with high purity and good integrity, facilitating subsequent experiments (Dong et al., 2012). After extracting the DNA, amplification is the next step. Researchers usually use polymerase chain reaction (PCR) to amplify certain regions of the chloroplast genome. In some experiments, especially when the initial amount of DNA is not large, a technique called “multi-primer loop amplification” is also used to enrich chloroplast DNA (Atherton et al., 2010). This method can not only increase the quantity of chloroplast DNA, but also improve its quality, which is beneficial to the subsequent high-throughput sequencing. 5.2 Sequencing techniques High-throughput sequencing platforms such as Illumina Genome Analyzer II (GAII) have been successfully applied in the sequencing of chloroplast DNA (Wang et al., 2010). This method can conduct comprehensive sequencing of chloroplast genomes. SNPs and Indels were identified from it (Figure 3) (Wang et al., 2018). There is also a technique called “genomic screening” (shallow sequencing of the entire genome), which can also obtain the complete chloroplast genome sequence. This method is highly efficient, not costly, and can also provide sufficient data for subsequent comparative genomic analysis. Figure 3 Conserved chloroplast genome structure inEucommia ulmoides (Adopted from Wang et al., 2018) Image caption: (A) Pairwise chloroplast genome alignments derived from Multiple Alignment using Fast Fourier Transform (MAFFT) program. The sequence identity is indicated on the top. Label KU204775.1 represents the E. ulmoides chloroplast genome retrieved from GenBank, while label E. ulmoides indicates the newly sequenced genome in this study. (B) Pairwise chloroplast genome alignments derived from MAUVE software (Adopted from Wang et al., 2018) Wang et al. (2018) discovered that the chloroplast genomic structure of Eucommia ulmoides was very stable. They compared the chloroplast genome sequence (labeled as KU204775.1) obtained from the GenBank database with the newly sequenced data using the MAFFT program and found that the sequences of the two were very similar, indicating that the internal variations of the chloroplast genome of Eucommia ulmoides are few and the structural changes are not significant. They also conducted further analysis using MAUVE software and found that the overall structure of the genome and the genetic content remained almost unchanged, proving its conservation.
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