Maize Genomics and Genetics 2024, Vol.15, No.4, 191-203 http://cropscipublisher.com/index.php/mgg 193 evolutionary history of Zea but also provide a framework for future studies on the genetic and functional diversity of plastid genomes in this important genus. 3 Methods and Techniques for Studying Changes in the Zea Plasmid Genome 3.1 Molecular biology techniques Molecular biology techniques are fundamental in studying the microstructural changes in the plastid genome of Zea species. One of the primary methods used is sequencing, which allows for the detailed examination of plastid genomes. In the study by Orton et al., 2017), both Sanger and next-generation sequencing methods were employed to sequence the complete plastomes of five Zea species. This comprehensive sequencing enabled the identification of 193 indels and 15 inversions, providing insights into the mutation rates and divergence times within the genus. Another critical molecular technique is the use of restriction endonucleases, which can compare the digestion patterns of affected and normal plastids. This method was utilized in the study of the iojap gene in Zea mays, which conditions a permanent deficiency in plastid differentiation. The restriction endonuclease digestion patterns confirmed that iojap-affected plastids contain a normal genome despite their inability to differentiate properly (Walbot and Coe, 1979). Additionally, the analysis of ribosomal internal transcribed spacer (ITS) sequences is a valuable molecular technique. ITS sequences were used to evaluate patterns of concerted evolution, rates of substitutions, and structural constraints in Zea and Tripsacum. This analysis revealed significant differences in ITS substitution rates among Zea taxa and identified methylation-induced deamination as a potent mutation source (Buckler and Holtsford, 1996). 3.2 Bioinformatics analysis Bioinformatics plays a crucial role in analyzing the vast amounts of data generated by sequencing technologies. Comparative genomic analysis is one such bioinformatics approach, which was used to study the plastomes of seven Lonicera species. This analysis identified various repeat sequence variations and protein sequence evolution, highlighting divergence hotspot regions and genes under positive selection (Liu et al., 2018). Phylogenomic analyses are another essential bioinformatics tool. In the study of Zea species, full plastome alignments were used to compare tree topologies from different types of mutations. This approach confirmed previous work examining Zea mitochondrial and nuclear data, providing a comprehensive view of the evolutionary relationships within the genus (Orton et al., 2017). Furthermore, the selection of appropriate evolutionary models and character partition strategies is critical for accurate phylogenetic analyses. The study on Amphilophium species demonstrated the importance of these models in recovering phylogenetic relationships and addressing sources of systematic error, such as compositional heterogeneity and codon usage bias (Thode et al., 2020). 3.3 Experimental and observational methods Experimental and observational methods are indispensable for understanding the functional implications of microstructural changes in the plastid genome. For instance, the effect of adenosine 5'-triphosphate (ATP) on the Shibata shift and associated structural changes in maize etioplasts was investigated through controlled experiments. The study found that ATP inhibits the Shibata shift and prolamellar body transformation, suggesting a regulatory role for ATP in early plastid development (Horton and Leech, 1975). Another experimental approach involves the reconstruction of gene transfer events from plastids to the nucleus. This method allows researchers to observe genome evolution in real-time and study the molecular mechanisms by which plastid genes are converted into functional nuclear genes. Such experiments have provided valuable insights into the ongoing process of DNA transfer from organelles to the nucleus (Bock and Timmis, 2008).
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