MGG_2024v15n5

Maize Genomics and Genetics 2024, Vol.15 http://cropscipublisher.com/index.php/mgg © 2024 CropSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved.

Maize Genomics and Genetics 2024, Vol.15 http://cropscipublisher.com/index.php/mgg © 2024 CropSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. CropSci Publisher is an international Open Access publishing specializing in maize genome, trait-controlling, maize gene expression and regulation at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada Publisher Cropsci Publisher Editedby Editorial Team of Maize Genomics and Genetics Email: edit@mgg.cropscipublisher.com Website: http://cropscipublisher.com/index.php/mgg Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Maize Genomics and Genetics (ISSN 1925-1971) is an open access, peer reviewed journal published online by CropSci Publisher. The journal is committed to publishing basic theories, novel techniques, and new advances within all aspects of maize research, especially focusing on genetics and genomics. Papers regarding classical genetics analysis, structural and functional analysis of maize genome, trait-controlling, maize gene expression and regulation, transgenic maize, as well as maize varietal improvement, are especially welcomed. All the articles published in Maize Genomics and Genetics are Open Access, and are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CropSci Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.

Maize Genomics and Genetics (online), 2024, Vol. 15, No.5 ISSN 1925-1971 http://cropscipublisher.com/index.php/mgg © 2024 CropSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Latest Content Phylogenomic Studies in Zea: Evolutionary Relationships and Species Divergence Jiansheng Li Maize Genomics and Genetics, 2024, Vol. 15, No. 5, 218-227 Genetic Diversity in the Genus Zea: Insights from Chloroplast Genome Variability Bin Chen, Junfeng Hou, Yunfei Cai, Guiyue Wang, Renxiang Cai, Fucheng Zhao Maize Genomics and Genetics, 2024, Vol. 15, No. 5, 228-238 Comparative Genomics of Maize: Insights into Evolution and Function Jin Zhou, Minli Xu Maize Genomics and Genetics, 2024, Vol. 15, No. 5, 239-246 High-Throughput Sequencing in Maize: A Gateway to Precision Breeding Lan Zhou, Long Jiang Maize Genomics and Genetics, 2024, Vol. 15, No. 5, 247-256 Trends in Maize Genomic Research: Past, Present, and Future Jinhua Cheng, Wei Wang Maize Genomics and Genetics, 2024, Vol. 15, No. 5, 257-269

Maize Genomics and Genetics 2024, Vol.15, No.5, 218-227 http://cropscipublisher.com/index.php/mgg 218 Systematic Review Open Access Phylogenomic Studies in Zea: Evolutionary Relationships and Species Divergence Jiansheng Li Sanya Institute of China Agricultural University, Sanya, 572025, Hainan, China Corresponding email: lijiansheng@cau.edu.cn Maize Genomics and Genetics, 2024, Vol.15, No.5 doi: 10.5376/mgg.2024.15.0021 Received: 08 Jul., 2024 Accepted: 15 Aug., 2024 Published: 06 Sep., 2024 Copyright © 2024 Li, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Li J.S., 2024, Phylogenomic studies in zea: evolutionary relationships and species divergence, Maize Genomics and Genetics, 15(5): 218-227 (doi: 10.5376/mgg.2024.15.0021) Abstract Phylogenomic studies have significantly advanced our understanding of the evolutionary relationships and species divergence within the genus Zea. By analyzing complete plastid genomes from five Zea species (Zea diploperennis, Zea perennis, Zea luxurians, Zea nicaraguensis, and Zea mays subsp. huehuetenangensis), this study investigates the rates and patterns of microstructural changes, including inversions and insertion/deletion mutations (indels). The findings reveal 193 indels and 15 inversions, with tandem repeat indels being the most prevalent. Divergence times were estimated using a noncorrelated relaxed clock method, indicating that the stem lineage of all Zea species diverged approximately 176 000 years before present (YBP). The mutation rates for the genus ranged from 1.7E-8 to 3.5E-8 microstructural changes per site per year, highlighting non-uniform rates of change despite close taxonomic relationships. These results corroborate previous studies on Zea mitochondrial and nuclear data, providing a comprehensive phylogenomic framework for understanding the evolutionary history and diversification of Zeaspecies. Keywords Maize (Zeamays); Phylogenomics; Zeaspecies; Microstructural changes; Divergence times; Plastid genomes 1 Introduction The genus Zea, belonging to the grass family Poaceae, is of significant agronomic and scientific importance. It includes several species, with maize (Zeamays) being the most prominent. Maize is not only a staple food crop but also a model organism for genetic and genomic research due to its extensive genetic diversity and well-characterized genome (Strable and Scanlon, 2009; Vincent, 2012). The genus Zea also includes wild relatives known as teosintes, which are crucial for understanding the evolutionary history and domestication of maize (Dermastia et al., 2009; Hufford et al., 2012a). The evolutionary study of Zea provides insights into plant domestication, adaptation, and the genetic basis of important agronomic traits (Kellogg and Birchler, 1993; Hilton and Gaut, 1998). Phylogenomics, the intersection of phylogenetics and genomics, is a powerful tool in evolutionary biology. It involves the analysis of genome-wide data to infer evolutionary relationships and understand the genetic basis of phenotypic diversity (Kellogg and Birchler, 1993). In the context of Zea, phylogenomic studies have elucidated the evolutionary relationships among different species and subspecies, shedding light on the processes of speciation and domestication (Hilton and Gaut, 1998; Ross-Ibarra et al., 2009). These studies are crucial for understanding the genetic mechanisms underlying important traits such as disease resistance, yield, and environmental adaptability (Strable and Scanlon, 2009; Mounika et al., 2018). By integrating phylogenetic and genomic data, researchers can gain a comprehensive understanding of plant evolution and the factors driving genetic diversity (Kellogg and Birchler, 1993; Curry 2020). This study investigates the evolutionary relationships within the genus Zea using phylogenomic approaches. This includes understanding the divergence times, gene flow, and adaptive evolution among different Zea species and subspecies. The study aims to contribute to the broader field of plant science by providing insights into the genetic basis of important agronomic traits and the evolutionary processes shaping plant diversity. Additionally, this research will inform conservation strategies for wild relatives of maize and guide breeding programs aimed at improving crop resilience and productivity.

Maize Genomics and Genetics 2024, Vol.15, No.5, 218-227 http://cropscipublisher.com/index.php/mgg 219 2 Methodological Approaches in Phylogenomics 2.1 Genomic data acquisition In phylogenomic studies of the genus Zea, various types of genomic data are utilized, including whole genome sequences and transcriptomic data. Whole genome sequencing, such as the sequencing of complete plastid genomes (plastomes), provides comprehensive data that can be used to analyze microstructural changes like inversions and indels (Orton et al., 2017) Transcriptomics, which involves sequencing RNA to study gene expression, is another valuable source of data. For instance, RNA sequencing has been used to assemble large and accurate phylogenomic datasets, as demonstrated in studies of jawed vertebrates (Irisarri et al., 2017). The methods for collecting and curating genomic data are diverse. Whole genome sequencing can be performed using next-generation sequencing technologies, which allow for the sequencing of entire genomes at a relatively low cost (Allio et al., 2019). Transcriptomic data can be obtained through RNA sequencing, which involves extracting RNA, converting it to cDNA, and then sequencing the cDNA (Irisarri et al., 2017). Data curation involves filtering and assembling the raw sequence data to ensure accuracy and completeness. For example, in the study of swallowtail butterflies, orthologous coding sequences were identified from whole-genome shotgun sequences, and these sequences were then used for phylogenomic analyses (Allio et al., 2019). 2.2 Phylogenomic analysis techniques Phylogenomic analysis involves several bioinformatics tools and techniques for alignment and phylogenetic tree construction. Tools such as IQ-TREE and PhyloBayes are commonly used for maximum-likelihood and Bayesian mixture model analyses, respectively (Allio et al., 2019). These tools help in constructing phylogenetic trees by aligning sequences and estimating evolutionary relationships based on the aligned data. Models of molecular evolution are crucial in phylogenomic analyses. These models describe how sequences evolve over time and are used to infer phylogenetic relationships. For instance, the noncorrelated relaxed clock method is used to estimate divergence times by allowing different parts of the genome to evolve at different rates (Orton et al., 2017). Other models, such as the uncorrelated lognormal (UCLN) model, are used to account for rate heterogeneity across genes and lineages (Smith et al., 2018). 2.3 Addressing methodological challenges Phylogenomic studies face several methodological challenges, including incomplete lineage sorting and hybridization. Incomplete lineage sorting occurs when the gene tree does not match the species tree due to ancestral polymorphisms. This issue can be addressed using multispecies coalescent methods, which consider the coalescent process of gene lineages within species (Koenen et al., 2019). Hybridization, which involves the mixing of genetic material from different species, can confound phylogenetic analyses. Techniques such as the D-statistic (ABBA-BABA test) are used to detect introgression and hybridization events (Vargas et al., 2017). Managing large genomic datasets is another significant challenge. Phylogenomic datasets can be vast, making it difficult to perform analyses on the entire dataset. One approach to manage this issue is "gene shopping," where a subset of genes with desirable properties (e.g., clock-likeness, reasonable tree length, and minimal topological conflict) is selected for analysis (Smith et al., 2018). This method helps reduce errors associated with model mis-specification and makes divergence-time estimation more efficient. 3 Evolutionary Relationships withinZea 3.1 Phylogenetic trees of Zea species The construction and interpretation of phylogenetic trees are fundamental to understanding the evolutionary relationships within the genus Zea. Phylogenetic trees are graphical representations that depict the evolutionary pathways and relationships among different species based on genetic data. In the context of Zea, complete plastid genomes (plastomes) have been utilized to construct these trees, providing insights into the microstructural changes such as inversions and insertion or deletion mutations (indels) that have occurred over time (Orton et al., 2017).

Maize Genomics and Genetics 2024, Vol.15, No.5, 218-227 http://cropscipublisher.com/index.php/mgg 220 The major evolutionary relationships revealed among Zea species indicate that despite the close genetic relationships, there are significant variations in mutation rates. For instance, the study of five Zea species, including Zea diploperennis, Zea perennis, Zea luxurians, Zea nicaraguensis, and Zea mays subsp. huehuetenangensis, showed that tandem repeat indels were the most common type of microstructural change observed. These findings are consistent with previous studies that have examined mitochondrial and nuclear data, confirming the robustness of the phylogenetic trees constructed using plastome alignments (Orton et al., 2017). 3.2 Divergence time estimation Estimating divergence times within the Zea genus involves sophisticated methods that model rate variation among lineages. One effective approach is the noncorrelated relaxed clock method, which allows for the estimation of divergence dates without assuming a constant rate of evolution across all branches of the phylogenetic tree. This method has been applied to calculate divergence times for specific nodes within Zea, revealing that the stem lineage of all Zea species diverged approximately 176 000 years before present (YBP) (Orton et al., 2017). Key divergence events within the Zea genus include the separation of subspecies around 38 000 YBP and the divergence of section Luxuriantes around 23 000 YBP. These temporal contexts provide a framework for understanding the evolutionary history and speciation events within the genus. The calculated mutation rates for Zea, ranging from 1.7E-8 to 3.5E-8 microstructural changes per site per year, highlight the non-uniformity of evolutionary rates despite the close relationships among taxa (Orton et al., 2017). 3.3 Evolutionary history of major Zea species The evolutionary pathways of major Zea species, such as maize (Zea mays), are shaped by a combination of genetic, environmental, and ecological factors. Maize, in particular, has undergone significant evolutionary changes that have been well-documented through phylogenomic studies. The use of complete plastid genomes has provided a detailed understanding of the microstructural changes and divergence times within the genus (Figure 1) (Orton et al., 2017). Figure 1 Plastid genome of Zeamays subsp. mays cv. ‘INIA 601’(Adopted from Orton et al., 2017) Image caption: The thick lines indicate the IR1 and IR2 regions, which separate the SSC and LSC regions. Genes inside the circle are transcribed in the clockwise direction and genes outside the circle in the counterclockwise direction. Colors of genes indicate their function as shown in the legend. Genes containing introns are marked with an asterisk (*) (Adopted from Orton et al., 2017) Factors influencing speciation within Zea include genetic mutations, environmental pressures, and hybridization events. The study of plastid genomes has revealed that tandem repeat indels are a common type of mutation,

Maize Genomics and Genetics 2024, Vol.15, No.5, 218-227 http://cropscipublisher.com/index.php/mgg 221 suggesting that these genetic changes play a crucial role in the evolutionary divergence of Zea species. Additionally, the non-uniform mutation rates observed across different species indicate that evolutionary pressures may vary significantly within the genus (Orton et al., 2017). 4Zea Species Divergence and Genetic Adaptation 4.1 Genetic differentiation among Zea species Genetic differentiation among Zea species is a complex process influenced by various evolutionary forces, including gene flow, natural selection, and genetic drift. Studies have shown that divergence in Zea species, such as Zeamays ssp. parviglumis and Zeamays ssp. mexicana, has occurred despite continuous gene flow, suggesting that local adaptation plays a significant role in maintaining species boundaries. The identification of key genes involved in species differentiation has been facilitated by genome-wide scans, which have revealed significant SNP associations with environmental variables like temperature and soil phosphorus concentration (Aguirre-Liguori et al., 2019). These findings indicate that specific genomic regions, possibly including putative inversions, contribute to reduced gene flow and increased genetic differentiation between locally adapted populations. In addition to local adaptation, historical gene flow has also played a crucial role in the divergence of Zea species. For instance, sequence polymorphism data from 26 nuclear loci have provided evidence for adaptive and purifying selection at nonsynonymous sites, highlighting the role of gene flow in the evolutionary history of Zea mays ssp. mays and three wild Zea taxa. This study estimated divergence times and suggested rapid diversification of lineages within Zea in the last~150 000 years, further emphasizing the importance of gene flow in shaping genetic differentiation (Ross-Ibarra et al., 2009). 4.2 Natural selection and adaptive traits Natural selection is a driving force in shaping adaptive traits in Zea species. The role of natural selection in adaptive divergence is evident from studies on other taxa, such as Acrossocheilus, where positive selection on mitochondrial genes has been linked to adaptation to different habitats (Zhao et al., 2022). Similarly, in Zea species, natural selection has likely played a crucial role in the evolution of adaptive traits that confer ecological advantages in specific environments. Case studies of adaptive traits in Zea species have highlighted the importance of environmental factors in shaping genetic variation. For example, the divergence between Zea mays ssp. parviglumis and Zea mays ssp. mexicana has been associated with adaptation to temperature and soil phosphorus concentration. Genome-wide scans have identified outlier SNPs linked to these environmental variables, suggesting that natural selection has targeted specific genomic regions to drive adaptive divergence (Aguirre-Liguori et al., 2019). Moreover, the study of genetic differentiation in other species, such as Daphnia pulex, has shown that regions of high gene density and recombination are more divergent, indicating that selection on genes related to local adaptation shapes genome-wide patterns of differentiation (Wersebe et al., 2022). These findings underscore the role of natural selection in driving adaptive traits and genetic differentiation in Zea species. 4.3 Hybridization and its evolutionary Impact Hybridization has played a significant role in the evolution of Zea species, contributing to genetic diversity and adaptive potential. Gene flow between hybridizing taxa can lead to heterogeneous genomic divergence, as observed in the teosinte subspecies Zeamays ssp. parviglumis and Zeamays ssp. mexicana. The continuous gene flow and secondary contact between these subspecies have resulted in genomic regions of high differentiation, likely driven by adaptive divergence and reduced gene flow in locally adapted populations (Aguirre-Liguori et al., 2019). Historical gene flow has also been a key factor in the divergence of Zea species. For instance, cultivated maize (Zea mays ssp. mays) may serve as a bridge for gene flow among otherwise allopatric wild taxa, facilitating the exchange of genetic material and promoting genetic diversity. This historical gene flow has likely contributed to

Maize Genomics and Genetics 2024, Vol.15, No.5, 218-227 http://cropscipublisher.com/index.php/mgg 222 the rapid diversification of lineages within Zea, as evidenced by the consistent estimates of divergence times from various markers (Ross-Ibarra et al., 2009). Case examples of gene flow between Zea species and its consequences can be seen in the study of pea aphid host races, where genomic hotspots of differentiation have been identified in regions associated with reproductive isolation and host-plant specialization (Nouhaud et al., 2018). These findings suggest that hybridization and gene flow can lead to the emergence of new adaptive traits and contribute to the evolutionary dynamics of Zea species. 5 Implications for Crop Improvement 5.1 Contributions to maize breeding programs The application of phylogenomic insights has significantly enhanced maize breeding programs by providing a deeper understanding of the genetic basis of important traits. For instance, the comprehensive assessment of maize evolution through genome-wide resequencing has identified numerous genes with strong signals of selection, which are crucial for major morphological changes in maize (Figure 2) (Hufford et al., 2012b). This information is invaluable for breeders aiming to develop new varieties with improved traits. Figure 2 Neighbor-joining tree and changing morphology of domesticated maize and its wild relatives (Adopted from Hufford et al., 2012b) Image caption: Taxa in the neighbor-joining tree (right) are represented by different colors: parviglumis (green), landraces (red), improved lines (blue), mexicana (yellow), and Tripsacum (brown). Morphological changes (left) are shown for female inflorescences and plant architecture during domestication and improvement (Adopted from Hufford et al., 2012b) Moreover, the integration of genomic prediction methods, such as GBLUP and BayesB, has facilitated more accurate predictions of hybrid performance, thereby optimizing the selection process in breeding programs (Technow et al., 2014). This approach allows breeders to focus on the performance of experimental hybrids rather than solely on parental lines, enhancing the efficiency of breeding strategies. Additionally, the identification of specific genetic loci associated with ear traits through GWAS and QTL mapping has provided targets for improving maize yield (Dong et al., 2023). By leveraging these genetic insights, breeders

Maize Genomics and Genetics 2024, Vol.15, No.5, 218-227 http://cropscipublisher.com/index.php/mgg 223 can develop maize varieties with enhanced ear traits, contributing to higher productivity. 5.2 Conservation of wild relatives Conserving the genetic diversity of wild Zea species is crucial for maintaining a reservoir of beneficial traits that can be integrated into cultivated maize. The domestication of maize from its wild ancestor, teosinte, involved significant metabolic divergence, with distinct sets of metabolites being targeted during different stages of maize evolution (Xu et al., 2019). This highlights the importance of preserving wild relatives to ensure the availability of diverse genetic resources. Strategies for integrating wild genetic resources into crop breeding include the use of genomic screening to identify genes affected by artificial selection during domestication and improvement (Yamasaki et al., 2007). By understanding the genetic basis of traits in wild relatives, breeders can introgress these beneficial alleles into cultivated maize, enhancing its adaptability and resilience. Furthermore, the use of temperate germplasm to improve tropical germplasm has shown potential in enhancing heterosis in grain yields (Wen et al., 2012). This approach involves incorporating unique alleles from temperate lines into tropically adapted lines, thereby increasing genetic diversity and improving crop performance. 5.3 Utilizing genetic diversity for agriculture Leveraging phylogenomic data to identify beneficial traits is a key strategy for improving agricultural productivity. For example, the identification of candidate genes contributing to metabolic divergence between maize and teosinte has provided insights into domestication-associated changes in metabolism (Xu et al., 2019). These findings can be used to develop maize varieties with enhanced metabolic traits, improving their nutritional quality and stress tolerance. Practical applications of phylogenomic findings in agriculture include the development of novel structural features in maize plants to increase yield and adaptability (Li et al., 2021). By understanding the phenotypic trait panorama, breeders can select for traits that enhance the structural efficiency of maize, such as improved nutrient transfer and epigenetic memory. Additionally, the integration of digital technologies and physiological knowledge into breeding programs has shown promise in hastening genetic gain (Diepenbrock et al., 2021). By combining crop growth models with whole genome prediction, breeders can more accurately predict the performance of untested genotypes in diverse environments, thereby accelerating the development of high-yielding maize varieties. 6 Future Directions inZea Phylogenomics 6.1 Advances in genomic technologies The advent of cutting-edge genomic technologies such as CRISPR and long-read sequencing has revolutionized the field of phylogenomics, offering unprecedented opportunities to delve deeper into the evolutionary relationships and species divergence within the genus Zea. Long-read sequencing technologies, for instance, have significantly enhanced our ability to sequence entire genomes with high accuracy, thereby providing a more comprehensive understanding of genetic variations and evolutionary patterns (McKain et al., 2018; Koenen et al., 2019; Guo et al., 2022). These technologies facilitate the identification of orthologous genes and the construction of more accurate phylogenetic trees, which are crucial for resolving complex evolutionary histories (Delsuc et al., 2005; Allio et al., 2019). CRISPR technology, on the other hand, offers the potential to manipulate specific genes and observe the resultant phenotypic changes, thereby providing insights into gene function and evolutionary adaptations (Guo et al., 2022; Chen et al., 2024). The integration of CRISPR with phylogenomic studies could enable researchers to experimentally validate hypotheses about gene function and evolutionary processes, thus bridging the gap between genomics and functional biology.

Maize Genomics and Genetics 2024, Vol.15, No.5, 218-227 http://cropscipublisher.com/index.php/mgg 224 Future prospects for using these technologies in Zea studies are promising. For instance, long-read sequencing could be employed to sequence the genomes of lesser-studied Zea species, thereby filling gaps in our current genomic databases and providing a more complete picture of the genus's evolutionary history (Orton et al., 2017). Additionally, CRISPR could be used to investigate the functional roles of specific genes identified through phylogenomic analyses, thereby enhancing researchers understanding of the genetic basis of key traits such as drought tolerance and disease resistance. 6.2 Integrating phylogenomics with other disciplines The integration of phylogenomics with other scientific disciplines such as ecology, physiology, and environmental science holds great potential for advancing researchers understanding of Zea evolution. By combining genomic data with ecological and physiological information, researchers can gain a more holistic view of how environmental factors and physiological adaptations have shaped the evolutionary trajectories of Zea species (Siepel, 2009; Fu et al., 2024). For example, ecological data on habitat preferences and environmental conditions can be used to contextualize phylogenomic findings, thereby providing insights into how different Zea species have adapted to their respective environments. Similarly, physiological studies on traits such as photosynthetic efficiency and water use can help elucidate the genetic basis of these adaptations and their evolutionary significance (Koenen et al., 2019). An interdisciplinary approach can also facilitate the identification of key genes and pathways involved in important traits, thereby informing breeding programs aimed at improving crop resilience and productivity. For instance, integrating phylogenomic data with physiological studies on drought tolerance could help identify candidate genes for genetic improvement, thereby contributing to the development of more resilient Zea varieties (Orton et al., 2017; McKain et al., 2018). 6.3 Addressing knowledge gaps Despite significant advances in Zea phylogenomics, several knowledge gaps remain. One major gap is the limited genomic data available for many Zea species, particularly those that are less economically important than Zea mays. This lack of data hampers researchers ability to construct comprehensive phylogenies and understand the full extent of genetic diversity within the genus (Orton et al., 2017; McKain et al., 2018). Another gap is the incomplete understanding of the evolutionary processes that have shaped the genetic diversity of Zea species. For instance, the roles of hybridization, introgression, and incomplete lineage sorting in Zea evolution are not fully understood and warrant further investigation (Vargas et al., 2017; Guo et al., 2022). Addressing these gaps will require the application of advanced genomic technologies and interdisciplinary approaches, as well as the generation of new genomic data for under-studied species. Proposed research directions to fill these gaps include the sequencing of additional Zea genomes using long-read technologies, which will provide more complete and accurate genomic data for phylogenetic analyses (Orton et al., 2017; McKain et al., 2018). Additionally, studies investigating the roles of hybridization and introgression in Zea evolution could provide valuable insights into the genetic mechanisms underlying species divergence and adaptation (Vargas et al., 2017). 7 Concluding Remarks The phylogenomic studies within the genus Zea have provided significant insights into the evolutionary relationships and species divergence. The analysis of complete plastid genomes (plastomes) across five Zea species revealed substantial microstructural changes, including 193 indels and 15 inversions, with tandem repeat indels being the most common. Divergence times were estimated, indicating that the stem lineage of all Zea species diverged approximately 176 000 years before present (YBP), with more recent divergence events occurring between 38 000 and 23 000 YBP. Additionally, the study confirmed previous findings from mitochondrial and nuclear data, reinforcing the robustness of the phylogenomic approach. Another study highlighted the role of gene flow in the evolutionary history of Zea, showing that gene flow has been a significant

Maize Genomics and Genetics 2024, Vol.15, No.5, 218-227 http://cropscipublisher.com/index.php/mgg 225 factor in population divergence and speciation within the genus. This study also provided consistent estimates of divergence times, suggesting rapid diversification within the last 150 000 years. Building on these findings, several potential areas of investigation emerge. Further exploration of the rates and patterns of microstructural changes across a broader range of Zea species could provide deeper insights into the mechanisms driving genomic evolution in this genus. Additionally, investigating the role of gene flow in more detail, particularly how cultivated maize may facilitate gene flow among wild taxa, could enhance understanding of the evolutionary dynamics within Zea. Another promising area is the examination of adaptive and purifying selection at nonsynonymous sites across different Zea species, which could shed light on the selective pressures shaping the genomes of these plants. Integrating data from nuclear, mitochondrial, and plastid genomes in a comprehensive phylogenomic framework could offer a more holistic view of the evolutionary history and relationships within Zea. Continued research in Zea phylogenomics is crucial for several reasons. Understanding the evolutionary relationships and divergence within this genus not only provides insights into the history and adaptation of these species but also has practical implications for agriculture and conservation. As Zea mays (maize) is a staple crop worldwide, knowledge of its genetic diversity and evolutionary history can inform breeding programs aimed at improving crop resilience and productivity. Moreover, studying the wild relatives of maize can reveal valuable genetic resources that may be harnessed for crop improvement. The findings from phylogenomic studies also contribute to the broader understanding of plant evolution and the processes driving speciation and adaptation. Therefore, continued research in this field is essential for advancing both basic and applied plant sciences. Acknowledgments CropSci Publisher thanks the anonymous reviewers for their insightful comments and suggestions that greatly improved the manuscript. Conflict of Interest Disclosure The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. References Aguirre-Liguori J.A., Gaut B.S., Jaramillo‐Correa J.P., Tenaillon M.I., Montes-Hernández S., García-Oliva F., Hearne S.J., and Eguiarte L.E., 2019, Divergence with gene flow is driven by local adaptation to temperature and soil phosphorus concentration in teosinte subspecies (Zea mays parviglumis and Zeamays mexicana), Molecular Ecology, 28(11): 2814-2830. https://doi.org/10.1111/mec.15098 PMID: 30980686 Allio R., Scornavacca C., Benoit, N., Clamens, A.L., Sperling F.A., and Condamine F.L., 2019, Whole genome shotgun phylogenomics resolves the pattern and timing of swallowtail butterfly evolution, Systematic Biology, 69(1): 38-60. https://doi.org/10.1093/sysbio/syz030 PMID: 31062850 Chen X.H., Qi X.H., and Xu X.W., 2024, From ancestors to modern cultivars: tracing the origin, evolution, and genetic progress in cucurbitaceae, Molecular Plant Breeding, 15(3): 112-131. https://doi.org/10.5376/mpb.2024.15.0013 Curry H., 2020, Taxonomy, race science, and mexican maize, Isis, 112(1): 1-21. https://doi.org/10.1086/713819 Delsuc F., Brinkmann H., and Philippe H., 2005, Phylogenomics and the reconstruction of the tree of life, Nature Reviews Genetics, 6(5): 361-375. https://doi.org/10.1038/nrg1603 Dermastia M., Kladnik A., Koce J.D., and Chourey P., 2009, A cellular study of teosinte Zea mays subsp. parviglumis (Poaceae) caryopsis development showing several processes conserved in maize, American Journal of Botany, 96(10): 1798-807. https://doi.org/10.3732/ajb.0900059 PMID: 21622300 Diepenbrock C.H,, Tang T., Jines M., Technow F., Lira S., Podlich D., Cooper M., and Messina C., 2021, Can we harness digital technologies and physiology to hasten genetic gain in united states maize breeding?, Plant Physiology, 188(2): 1141-1157. https://doi.org/10.1093/plphys/kiab527. PMID: 34791474 PMCID: PMC8825268

Maize Genomics and Genetics 2024, Vol.15, No.5, 218-227 http://cropscipublisher.com/index.php/mgg 226 Dong Z.Y., Wang Y.B., Bao J.X., Li Y.N., Yin Z.C., Long Y., and Wan X.Y., 2023, The genetic structures and molecular mechanisms underlying ear traits in maize (Zeamays L.), Cells, 12(14): 1900. https://doi.org/10.3390/cells12141900 PMID: 37508564 PMCID: PMC10378120 Fu J., Hong Z.M., and Huang W.Z., 2024, Harnessing genomic tools for cassava improvement: advances and prospects, Bioscience Evidence, 14(1): 32-38. https://doi.org/10.5376/be.2024.14.0005 Guo C., Luo Y., Gao L.M., Yi T.S., Li H.T., Yang J.B., and Li D.Z., 2022, Phylogenomics and the flowering plant tree of life, Journal of Integrative Plant Biology, 65(2): 299-323. https://doi.org/10.1111/jipb.13415 PMID: 36416284 Hilton H., and Gaut B.S., 1998, Speciation and domestication in maize and its wild relatives: evidence from the globulin-1 gene, Genetics, 150(2): 863-872. https://doi.org/10.1093/genetics/150.2.863 PMID: 9755214 PMCID: PMC1460357 Hufford M.B., Martínez‐Meyer E., Gaut B.S., Eguiarte L.E., and Tenaillon M.I., 2012a, Inferences from the historical distribution of wild and domesticated maize provide ecological and evolutionary insight, PLoS One, 7(11): e47659. https://doi.org/10.1371/journal.pone.0047659 PMID: 23155371 PMCID: PMC3498274 Hufford M.B., Xu X., van Heerwaarden J., Pyhäjärvi T., Chia J.M., Cartwright R.A., Elshire R.J., Glaubitz J.C., Guill K.E., Kaeppler S.M., Lai J.S., Morrell P.L., Shannon L.M., Song C., Springer N.M., Swanson-Wagner R.A., Tiffin P., Wang J., Zhang G.Y., Doebley J., McMullen M., Ware D., Buckler E.S., Yang S., and Ross-Ibarra J., 2012b, Comparative population genomics of maize domestication and improvement, Nature Genetics, 44(7): 808-811. https://doi.org/10.1038/ng.2309 PMID: 22660546 PMCID: PMC5531767 Irisarri I., Baurain D., Brinkmann H., Delsuc F., Sire J.Y., Kupfer A., Petersen J., Jare M., Meyer A., Vences M., and Philippe H., 2017, Phylotranscriptomic consolidation of the jawed vertebrate timetree, Nature Ecology and Evolution, 1(9): 1370-1378. https://doi.org/10.1038/s41559-017-0240-5 PMID: 28890940 PMCID: PMC5584656 Kellogg E.A., and Birchler J.A., 1993, Linking phylogeny and genetics: Zeamays as a tool for phylogenetic studies, Systematic Biology, 42(4): 415-439. https://doi.org/10.1093/SYSBIO/42.4.415. Koenen E.J.M., Ojeda D.I., Steeves R., Migliore J., Bakker F.T., Wieringa, J.J., Kidner C., Hardy O.J., Pennington R.T., Bruneau A., and Hughes C.E., 2019, Large‐scale genomic sequence data resolve the deepest divergences in the legume phylogeny and support a near‐simultaneous evolutionary origin of all six subfamilies, The New Phytologist, 225(3): 1355-1369. https://doi.org/10.1111/nph.16290 PMID: 31665814 PMCID: PMC6972672 Li Q.L., Gao L., Liu D.F., Xu L., Zhang X.X., and Zhang C.Q., 2021, Novel insights of maize structural feature in China, Euphytica, 217(1): 1-17. https://doi.org/10.1007/s10681-020-02742-3 McKain M.R., Johnson M.G., Uribe-Convers S., Eaton D., and Yang Y., 2018, Practical considerations for plant phylogenomics, Applications in Plant Sciences, 6(3): e1038. https://doi.org/10.1002/aps3.1038. PMID: 29732268 PMCID: PMC5895195 Mounika K., Ahamed M.L., and Umar S.N., 2018, Principal component and cluster analysis in inbred lines of maize (Zea mays L.), International Journal of Current Microbiology and Applied Sciences, 7(6): 3221-3229. https://doi.org/10.20546/IJCMAS.2018.706.379 PMID: 27488804 Nouhaud P., Gautier M., Gouin A., Jaquiéry J., Peccoud J., Legeai F., Mieuzet L., Smadja C.M., Lemaitre C., Vitalis R., and Simon J., 2018, Identifying genomic hotspots of differentiation and candidate genes involved in the adaptive divergence of pea aphid host races, Molecular Ecology, 27: 3287-3300. https://doi.org/10.1111/mec.14799. Orton L.M., Burke S.V., Wysocki W.P., and Duvall M.R., 2017, Plastid phylogenomic study of species within the genus Zea: rates and patterns of three classes of microstructural changes, Current Genetics, 63: 311-323. https://doi.org/10.1007/s00294-016-0637-8 Ross-Ibarra J., Tenaillon M., and Gaut B.S., 2009, Historical divergence and gene flow in the genus Zea, Genetics, 181: 1399-1413. https://doi.org/10.1534/genetics.108.097238 PMID: 19153259 PMCID: PMC2666508 Siepel A., 2009, Phylogenomics of primates and their ancestral populations, Genome Research, 19(11): 1929-1941. https://doi.org/10.1101/gr.084228.108 PMID: 19801602 PMCID: PMC2775601 Smith S.A., Brown J.W., and Walker J.F., 2018, So many genes, so little time: a practical approach to divergence-time estimation in the genomic era, PLoS One, 13(5): e0197433. https://doi.org/10.1371/journal.pone.0197433

Maize Genomics and Genetics 2024, Vol.15, No.5, 218-227 http://cropscipublisher.com/index.php/mgg 227 Strable J., and Scanlon M.J., 2009, Maize (Zea mays): a model organism for basic and applied research in plant biology, Cold Spring Harbor Protocols, 2009(10): pdb.emo132. https://doi.org/10.1101/pdb.emo132 PMID: 20147033 Technow F., Schrag T.A., Schipprack W., Bauer E., Simianer H., and Melchinger A.E., 2014, Genome properties and prospects of genomic prediction of hybrid performance in a breeding program of maize, Genetics, 197: 1343-1355. https://doi.org/10.1534/genetics.114.165860. PMID: 24850820 PMCID: PMC4125404 Vargas O.M., Ortiz E.M., and Simpson B.B., 2017, Conflicting phylogenomic signals reveal a pattern of reticulate evolution in a recent high-Andean diversification (asteraceae: astereae: diplostephium), The New Phytologist, 214(4): 1736-1750. https://doi.org/10.1111/nph.14530 PMID: 28333396 Vincent P.L.D., 2012, ZeaMays (maize, corn), .eLS, (2012): e3687. https://doi.org/10.1002/9780470015902.A0003687 Wen W.W., Guo T.T., Tovar V.H.C., Li H.H., Yan J.B., and Taba S., 2012, The strategy and potential utilization of temperate germplasm for tropical germplasm improvement: a case study of maize (Zeamays L.), Molecular Breeding, 29: 951-962. https://doi.org/10.1007/s11032-011-9696-1 PMID: 35119153 Wersebe M.J., Sherman R.E., Jeyasingh P.D., and Weider L.J., 2022, The roles of recombination and selection in shaping genomic divergence in an incipient ecological species complex, Molecular Ecology, 32: 1478-1496. https://doi.org/10.1111/mec.16383 Xu G.H., Cao J.J., Wang X.F., Chen Q.Y., Jin W.W., Li Z., and Tian F., 2019, Evolutionary metabolomics identifies substantial metabolic divergence between maize and its wild ancestor, Teosinte, Plant Cell, 31(9): 1990-2009. https://doi.org/10.1105/tpc.19.00111 PMID: 31227559 PMCID: PMC6751114 Yamasaki M., Wright S.I., and McMullen M.D., 2007, Genomic screening for artificial selection during domestication and improvement in maize, Annals of Botany, 100(5): 967-973. https://doi.org/10.1093/aob/mcm173 PMID: 17704539 PMCID: PMC2759211 Zhao D., Guo Y.D., and Gao Y., 2022, Natural selection drives the evolution of mitogenomes in Acrossocheilus, PLoS One, 17(10): e0276056. https://doi.org/10.1371/journal.pone.0276056 PMID: 36227932 PMCID: PMC9560497

Maize Genomics and Genetics 2024, Vol.15, No.5, 228-238 http://cropscipublisher.com/index.php/mgg 228 Systematic Review Open Access Genetic Diversity in the Genus Zea: Insights from Chloroplast Genome Variability BinChen1, Junfeng Hou1, Yunfei Cai 2, Guiyue Wang1, Renxiang Cai 3, Fucheng Zhao1 1 Institute of Maize and Featured Upland Crops, Zhejiang Academy of Agricultural Sciences, Dongyang, 322100, Zhengjiang, China 2 Seed Management Station of Zhejiang Province, Hangzhou, 310009, Zhengjiang, China 3 Institute of Life Science, Jiyang College of Zhejiang AandF University, Zhuji, 311800, Zhengjiang, China Corresponding author: ymszfc@163.com Maize Genomics and Genetics, 2024, Vol.15, No.5 doi: 10.5376/mgg.2024.15.0022 Received: 22 Jul., 2024 Accepted: 03 Sep., 2024 Published: 23 Sep., 2024 Copyright © 2024 Chen et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Chen B., Hou J.F., Cai Y.F., Wang G.Y., Cai R.X., and Zhao F.C., 2024, Genetic diversity in the genus zea: insights from chloroplast genome variability, Maize Genomics and Genetics, 15(5): 228-238 (doi: 10.5376/mgg.2024.15.0022) Abstract The genus Zea, which includes maize and its wild relatives, exhibits significant genetic diversity, particularly within the chloroplast genome. This study investigates the variability in chloroplast genomes across different Zea species to understand their evolutionary relationships and potential for genetic improvement. By analyzing whole chloroplast genomes, researchers identified substantial nucleotide sequence variations and evolutionary rates among different Zea species. Our findings reveal that photosynthetic genes are under strong purifying selection, while other genes exhibit heterogeneous substitution rates, indicating diverse evolutionary pressures. This research highlights the importance of chloroplast genome diversity in the adaptation and evolution of Zea species, providing valuable insights for breeding programs aimed at enhancing stress tolerance and other agronomic traits. Keywords Genetic diversity; Chloroplast genome; Zeaspecies; Evolutionary rates; Plant breeding 1 Introduction The genus Zea, particularly Zea mays (maize), holds a pivotal role in global agriculture and nutrition. Maize is one of the world's most important cereal crops, extensively cultivated for its versatility in human food, animal feed, and industrial applications (Nuss and Tanumihardjo, 2010; Lee et al., 2019; Revilla et al., 2022). Originating from Central America, maize has adapted to diverse environments, resulting in a wide range of genetic resources with significant variability (Revilla et al., 2022). This adaptability has made maize a staple food for a substantial portion of the global population, particularly in regions such as sub-Saharan Africa, Southeast Asia, and Latin America, where it is a primary source of nutrition (Nuss and Tanumihardjo, 2010). The genetic diversity within maize is not only crucial for its adaptability and yield improvement but also for its nutritional enhancement through biofortification strategies (Nuss and Tanumihardjo, 2010; Dong et al., 2023). The chloroplast genome plays a critical role in plant phylogeny and evolutionary studies. Chloroplast DNA (cpDNA) is maternally inherited in most plant species and exhibits a relatively slow mutation rate compared to nuclear DNA, making it a valuable tool for studying evolutionary relationships and genetic diversity among plant species (Vivodík et al., 2017). In maize, the chloroplast genome has been utilized to explore genetic diversity and phylogenetic relationships, providing insights into the evolutionary history and domestication of this important crop (Strable and Scanlon, 2009; Vivodík et al., 2017). The cpDNA markers are particularly useful in assessing genetic variation and identifying distinct genetic lineages within the genus Zea, which can inform breeding programs and conservation efforts (Vivodík et al., 2017). This research is to investigate the genetic diversity within the genus Zea by analyzing chloroplast genome variability. This study aims to elucidate the phylogenetic relationships among different Zea species and subspecies, with a particular focus on Zea mays. By leveraging cpDNA markers, we seek to uncover the extent of genetic variation and identify distinct genetic lineages within the genus. This research will contribute to a deeper understanding of the evolutionary history and domestication processes of maize, providing valuable information for breeding programs aimed at improving crop yield, adaptability, and nutritional quality. Additionally, the

Maize Genomics and Genetics 2024, Vol.15, No.5, 228-238 http://cropscipublisher.com/index.php/mgg 229 findings will have implications for the conservation of genetic resources within the genus Zea, ensuring the sustainability and resilience of this vital crop in the face of environmental challenges. 2 Methods for Comparing Chloroplast Genes 2.1 Collection and analysis of chloroplast genome data To investigate the genetic diversity within the genus Zea, we collected chloroplast genome data from various sources. The primary data sources included newly sequenced genomes and publicly available sequences from databases such as GenBank. Sequencing technologies employed in these studies ranged from traditional Sanger sequencing to next-generation sequencing platforms like Illumina, which provide high-throughput and accurate genome sequences (Bayly et al., 2013; Li et al., 2020; Loeuille et al., 2021). The analysis of chloroplast genome variability involved several bioinformatics tools and methods. For instance, tools like GYDLE Inc. pipelines were used for direct chloroplast genome assembly, ensuring high-quality finished genomes without the need for PCR gap-filling or contig order resolution (Bayly et al., 2013). Comparative genomics tools such as CGView Comparison Tool (CCT) were utilized to compare chloroplast genomes across different species, identifying structural variations and sequence similarities (Gao et al., 2019). Additionally, software like MEGA and PAUP* were employed for phylogenetic analyses, while custom scripts in languages like Python and R were used for detailed sequence analysis and visualization (Doebley et al., 1987; Dong et al., 2012). 2.2 Genome alignment and variation detection Aligning chloroplast genome sequences is a critical step in identifying genetic variations. Multiple sequence alignment tools such as MAFFT and ClustalW were used to align the chloroplast genomes of Zea species and their relatives. These tools help in aligning sequences accurately, allowing for the detection of conserved and variable regions (Saski et al., 2007; Li et al., 2020; Loeuille et al., 2021). To identify and classify variations, several strategies were employed. Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were detected using tools like GATK and SAMtools, which provide robust methods for variant calling from aligned sequence data (Dong et al., 2012; Li et al., 2020). Additionally, regions with high nucleotide diversity were identified using sliding window analysis, which helps in pinpointing hotspots of genetic variation (Shaw et al., 2007; Xie et al., 2018). The identified variations were then annotated using databases like dbSNP and tools such as SnpEff, which provide functional insights into the detected variants (Doebley et al., 1987; Soltis et al., 1991). 2.3 Phylogenetic and population genetics analysis Phylogenetic tree construction is essential for understanding the evolutionary relationships among species. In this study, phylogenetic trees were constructed using maximum parsimony and Bayesian inference methods. Software like MrBayes and RAxML were employed to generate phylogenetic trees based on chloroplast genome sequences, providing insights into the interspecies relationships within the genus Zea (Saski et al., 2007; Bayly et al., 2013). These analyses revealed that chloroplast DNA data could produce trees consistent with other measures of species affinity, such as isoenzymatic and morphological data (Doebley et al., 1987). Population genetics analysis was conducted to study gene flow and population structure within Zea species. Tools like STRUCTURE and Arlequin were used to analyze genetic diversity and population structure, providing insights into the genetic differentiation and admixture among populations (Xie et al., 2018). These analyses were complemented by the use of highly variable chloroplast markers, which are particularly useful for evaluating phylogeny at low taxonomic levels and for DNA barcoding (Soltis et al., 1991; Dong et al., 2012). The combination of phylogenetic and population genetics analyses allowed for a comprehensive understanding of the genetic diversity and evolutionary history of the genus Zea. 3 Genetic Diversity inZea Revealed by Chloroplast Genome Variability 3.1 Structural variation in chloroplast genomes The chloroplast genomes of Zea species exhibit notable structural variations that contribute to our understanding of genetic diversity within this genus. Structural rearrangements, such as inversions and transpositions, have been

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