Molecular Plant Breeding 2025, Vol.16 http://genbreedpublisher.com/index.php/mpb © 2025 GenBreed Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Publisher
Molecular Plant Breeding 2025, Vol.16 http://genbreedpublisher.com/index.php/mpb © 2025 GenBreed Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. GenBreed Publisher is an international Open Access publisher specializing in molecular genetics, plant genes or traits, and plant breeding registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada. Publisher GenBreed Publisher Edited by Editorial Team of Molecular Plant Breeding Email: edit@mpb.genbreedpublisher.com Website: http://genbreedpublisher.com/index.php/mpb Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Molecular Plant Breeding (ISSN 1923-8266) is an international, open access, peer reviewed journal published online by GenBreed Publisher. The journal publishes all the latest and outstanding research articles, letters and reviews in all areas of transgene, molecular genetics, crop QTL analysis, germplasm genetic diversity, and advanced breeding technologies. Molecular Plant Breeding is archived in LAC (Library and Archives Canada) and deposited in CrossRef. The Journal has been indexed by ProQuest as well. The Journal is expected to be indexed by PubMed and other databases in near future. All the articles published in Molecular Plant Breeding 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. GenBreed Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.
Molecular Plant Breeding (online), 2025, Vol. 16, No.5 ISSN 1923-8266 http://genbreedpublisher.com/index.php/mpb © 2025 GenBreed Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher Latest Content Epigenetic Mechanisms Regulating Lycopene Accumulation in Solanum lycopersicum Jiong Fu, Zhongmei Hong Molecular Plant Breeding, 2025, Vol. 16, No. 5, 261-267 Haplotype-Based Breeding of Yield-Related Traits in Oryza sativa: From Genomic Insights to Field Applications Qifu Zhang, Ruchun Chen, Zhongxian Li, Jianquan Li Molecular Plant Breeding, 2025, Vol. 16, No. 5, 268-277 Molecular Breeding Strategies for Aroma and Flavor Enhancement in Camellia sinensis Jianli Lu, Xiaocheng Wang Molecular Plant Breeding, 2025, Vol. 16, No. 5, 278-286 Breeding Strategies of Ginkgo biloba for Medicinal and Ornamental Uses: Progress, Challenges, and Future Perspectives Xichen Wang, Jianmin Zheng, Chuchu Liu Molecular Plant Breeding, 2025, Vol. 16, No. 5, 287-293 Integrated Performance of Fertilization Regimes on Pungency and Disease Resistance in Hangjiao Pepper under Organic Cultivation Dandan Huang, Xignzhu Feng, Haimei Wang Molecular Plant Breeding, 2025, Vol. 16, No. 5, 294-302
Molecular Plant Breeding 2025, Vol.16, No.5, 261-267 http://genbreedpublisher.com/index.php/mpb 261 Research Insight Open Access Epigenetic Mechanisms Regulating Lycopene Accumulation in Solanum lycopersicum Jiong Fu , Zhongmei Hong Hainan Provincial Key Laboratory of Crop Molecular Breeding, Sanya, 572025, Hainan, China Corresponding email: jiong.fu@hibio.org Molecular Plant Breeding, 2025, Vol.16, No.5 doi: 10.5376/mpb.2025.16.0026 Received: 01 Aug., 2025 Accepted: 06 Sep., 2025 Published: 14 Sep., 2025 Copyright © 2025 Fu and Hong, 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: Fu J., and Hong Z.M., 2025, Epigenetic mechanisms regulating lycopene accumulation in Solanum lycopersicum, Molecular Plant Breeding, 16(5): 261-267 (doi: 10.5376/mpb.2025.16.0026) Abstract This study summarizes the role of epigenetic mechanisms in regulating lycopene accumulation in tomatoes, with a focus on DNA methylation, histone modification, and non-coding RNA, etc. It introduces the latest progress in their regulation of fruit development, ripening, and metabolism, and discusses the interactions between these epigenetic modifications and environmental factors as well as hormone signals. The potential mechanisms of their role in metabolic pathway regulation and fruit quality improvement were explained. By integrating new technologies such as high-throughput sequencing, multi-omics integration, and CRISPR/dCas9, the application prospects of epigenetic information in tomato molecular breeding, metabolic engineering, and precision agriculture were evaluated. This study aims to provide a theoretical basis and molecular strategies for increasing lycopene content and the nutritional quality of fruits. Keywords Tomato (Solanum lycopersicum); Lycopene; Epigenetic regulation; DNA methylation; Histone modifications 1 Introduction Lycopene is one of the most common carotenoids in the fruit of tomatoes (Solanum lycopersicum). It not only gives the fruit a bright red color, but also is regarded as very important for human health due to its strong antioxidant activity. As the main source of lycopene in human diet, it is closely related to health benefits such as reducing the risk of cardiovascular diseases and some cancers. The accumulation of lycopene is also a key process for fruit ripening and quality formation, which is regulated by many endogenous and exogenous factors (Luo et al., 2013; McQuinn et al., 2017; Ming et al., 2023). Epigenetics regulates gene expression through heritable means without altering the DNA sequence, including DNA methylation, histone modification, regulation of non-coding RNA, etc. (Wu and Li, 2024). Recent studies have found that these mechanisms play an important role in the development and ripening of tomato fruits. DNA methylation and histone deacetylation can affect the expression of genes related to carotenoid synthesis and regulate the accumulation of lycopene. The research by He et al. (2024) and Nazari et al. (2025) indicates that non-coding RNAs are involved in the regulatory network of fruit ripening and pigment accumulation. This study summarizes the epigenetic mechanisms regulating lycopene accumulation, introduces the roles of DNA methylation, histone modification, etc. in fruit development and ripening, and reveals how these mechanisms jointly regulate the expression of carotenoid synthesis genes by combining the latest molecular biology and epigenetic research. This study aims to provide a theoretical basis and new methods for precisely regulating lycopene content and improving fruit quality. 2 Lycopene Biosynthesis inSolanum lycopersicum 2.1 Carotenoid biosynthetic pathway: key enzymes and regulation points Lycopene belongs to the carotenoid family. Its synthetic pathway starts with isoprenyl pyrophosphate (IPP) and is completed through the action of a series of enzymes. The key enzymes include PSY1, PDS, ZDS, ZISO and CRTISO. PSY1 is a rate-limiting enzyme that controls the flow of the entire pathway. CRTISO converts the precursor prolycopene into lycopene. If this enzyme is lacking, prolycopene will accumulate and the fruit will turn orange instead of red (Zhang et al., 2018; Prashanth et al., 2023).
Molecular Plant Breeding 2025, Vol.16, No.5, 261-267 http://genbreedpublisher.com/index.php/mpb 262 2.2 Genetic determinants influencing lycopene content Many genes regulate the content of lycopene. In addition to these structural genes, the regulatory gene SlIPT4 (encoding isoprenyltransferase) can positively regulate lycopene synthesis by affecting the expression of ZISO, and has a feedback regulatory effect on ABA (abscisic acid) signaling (Zhang et al., 2018). In addition, the deletion of CRTISO gene function (such as the Tan406 mutant) can block the conversion of prolycopene to lycopene, altering the color and nutritional value of the fruit (Prashanth et al., 2023). Transgenic studies have found that exogenous expression of lycopene β-cyclase can significantly increase the content of β-carotene and enhance the flow of the entire carotenoid pathway (Apel and Bock, 2009). 2.3 Developmental and environmental factors affecting accumulation The accumulation of lycopene can also be influenced by the stage of fruit development and environmental conditions. When the fruit ripens, hormones such as ethylene and jasmonic acid (JA) regulate the expression of related genes and promote the synthesis of lycopene. JA can not only promote lycopene accumulation independently of ethylene, but also restore the lycopene content of JA-deficient mutants through exogenous treatment (Liu et al., 2012). Environmental stresses such as salt stress can inhibit photosynthesis, but at the same time induce the expression of key genes such as PSY1, PDS, ZDS, and LYCB, increase the accumulation of lycopene and other carotenoids, and this effect is specific in different varieties and developmental stages (Leiva-Ampuero et al., 2020). 3 Epigenetic Regulation of Lycopene Biosynthesis 3.1 DNA methylation and transcriptional control of carotenoid genes DNA methylation is an important mechanism regulating the ripening of tomato fruits and the expression of carotenoid synthesis genes. During the fruit ripening process, the methylation levels in the promoter regions of key carotenoid synthesis genes (such as PSY1, PDS, etc.) undergo dynamic changes, which affect their transcriptional activity and regulate the synthesis and accumulation of lycopene (Ming et al., 2023). Nazari et al. (2025) found that external stresses, such as nanoplastic contamination, can also alter DNA methylation status and indirectly affect lycopene content. DNA methylation also interacts with transcription factors and ethylene signaling pathways to form a complex regulatory network (McQuinn et al., 2017). 3.2 Histone modifications and chromatin remodeling in carotenoid regulation Histone modification is also crucial in regulating the gene expression of fruit ripening and carotenoid synthesis. After histone deacetylases (such as SlHDA1, SlHDA3, etc.) are silenced, the fruit will ripen more quickly. Histone variants (such as Sl_H2A.Z) directly regulate the expression of key genes such as PSY1 and PDS, and affect the synthesis of lycopene (Figure 1) (Ming et al., 2023; Nazari et al., 2025). Ming et al. (2023) hold that these modifications alter chromatin structure and influence gene accessibility and transcriptional activity, and are one of the core epigenetic mechanisms regulating lycopene accumulation. 3.3 Role of microRNAs, lncRNAs, and other non-coding RNAs in post-transcriptional regulation Non-coding RNAs, especially microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), also play a significant role in the post-transcriptional regulation of fruit ripening and lycopene accumulation. High-throughput sequencing has identified a variety of miRNAs and lncRNAs related to fruit ripening, which affect lycopene accumulation by regulating the mRNA stability and translation efficiency of carotenoid synthesis genes (Ming et al., 2023). In their 2018 study, Chen et al. found that miR1916 can regulate the expression of transcription factors and related enzymes, indirectly affecting the accumulation of secondary metabolites, indicating that miRNA may play a key role in regulating the synthesis of lycopene and other pigments. 4 Interaction Between Epigenetic Modifications and Environmental Cues 4.1 Temperature, light, and stress effects on epigenetic regulation Environmental factors can affect the ripening of tomato fruits and the accumulation of lycopene through epigenetic mechanisms. Light regulates the synthesis of lycopene through phytochromes in fruits. Alba et al. (2000) found in their early research that red light treatment could significantly promote the accumulation of lycopene in tomato fruits, while far-red light would reverse this effect, indicating that photosensitive pigments are
Molecular Plant Breeding 2025, Vol.16, No.5, 261-267 http://genbreedpublisher.com/index.php/mpb 263 crucial in the light-induced accumulation of lycopene, and this process does not rely on the production of ethylene. Environmental stress can alter DNA methylation levels by up-regulating epigenetic genes such as histone deacetylase (HDA3), and affect fruit development, ripening and lycopene content (Nazari et al., 2025). Figure 1 The regulation network of DNA methylation, RNA methylation, histone modification and non-coding RNA in tomato fruit ripening (Adopted from Ming et al., 2023) Image caption: SlALKBH2 regulates the stability of SlDML2 RNAby RNAm6A demethylation, enhancing its stability. Conversely, SlDML2 promotes the expression of SlALKBH2 through DNA demethylation, ultimately affecting fruit ripening. The histone demethylase SlJMJ6 eliminates H3K27me marks fromSlDML2, RIN and ripening-associated genes, consequently promoting fruit ripening. On the other hand, histone demethylase SlJMJ7 erases H3K4me modifications from these genes, thus contributing to the inhibition of fruit ripening. Tomato methyltransferase SlMET1, Histone deacetylases SlHDA1, SlHDA3 and SlHDT1 may negatively regulate ripening-associated genes to control fruit ripening, while SlHDT3 exhibits opposing regulatory effects. PRC1 protein SlLHP1b could bind H3K27me mark in regions of ripening-associated chromatin, targeting ripening-related genes, repressing fruit ripening. Meanwhile, SlLHP1b interacted with PRC2 protein SlMSI1, negatively regulating tomato fruit ripening. The microRNA gene SlMIR164Awas involved in negative regulation of tomato fruit ripening (Adopted from Ming et al., 2023) 4.2 Hormonal regulation (ethylene, ABA) mediated by epigenetic mechanisms Ethylene is the main hormone regulating the ripening of tomato fruits and the accumulation of lycopene. Epigenetic mechanisms are closely related to the ethylene signaling pathway. Studies have found that the silencing of histone deacetylases (such as SlHDA1, SlHDA3, etc.) will accelerate fruit ripening, while their upregulation will delay ripening and indirectly affect the accumulation of lycopene (Nazari et al., 2025). Ming et al. (2023) demonstrated in their research that hormones such as abscisic acid (ABA) may also influence the expression of related genes by regulating epigenetic modifications, but the specific mechanisms still require further study. 4.3 Crosstalk between metabolic pathways and epigenetic regulation The biosynthesis of lycopene involves multiple metabolic pathways, and the expression of key enzyme genes in these pathways is regulated by epigenetic modifications. The histone variant Sl_H2A.Z regulates the expression of genes related to carotenoid biosynthesis (such as SlPSY1, SlPDS, etc.) and affects lycopene accumulation (Ming et al., 2023). Non-coding RNAs are involved in the regulation of fruit ripening and pigment accumulation by targeting key genes in metabolic pathways (Chen et al., 2018; Ming et al., 2023). The interaction between epigenetic mechanisms and metabolic networks together constitutes a complex system for regulating lycopene accumulation.
Molecular Plant Breeding 2025, Vol.16, No.5, 261-267 http://genbreedpublisher.com/index.php/mpb 264 5 Case Study: Epigenetic Mechanisms in High-Lycopene Tomato Cultivars 5.1 Selection of contrasting tomato cultivars for analysis When studying the epigenetic regulation of lycopene accumulation, comparison varieties with high lycopene and low lycopene are usually selected. These varieties have significant differences in lycopene content during the fruit ripening process and are very suitable for studying the differences in epigenetic regulation. The commonly used varieties include ‘MicroTom’, ‘Ailsa Craig’ and their mutants. The lycopene content and the expression of related genes of these varieties show significant differences at the fruit development and ripening stages (Ming et al., 2023; He et al., 2024). 5.2 Experimental methods: methylation mapping, transcriptome profiling To systematically study epigenetic mechanisms, researchers usually employ high-throughput technologies such as whole-genome DNA methylation sequencing (WGBS) and transcriptome sequencing (RNA-seq). Methylation profiling analysis can determine the methylation status of the promoter regions of genes related to lycopene synthesis and reveal the regulatory role of DNA methylation on gene expression. Transcriptome analysis is used to compare the expression levels of key genes in the lycopene synthesis pathway under different varieties or different treatment conditions. Histone modifications and the expression of non-coding RNAs were also detected by methods such as ChIP-seq and small RNA sequencing (Ming et al., 2023; He et al., 2024). 5.3 Key findings on epigenetic marks correlating with lycopene levels Research has found that in high-lycopene varieties, the DNA methylation levels in the promoter regions of key lycopene synthesis genes (such as PSY1, PDS, etc.) are significantly reduced. This will enable these genes to be expressed at high levels, thereby promoting the accumulation of lycopene. In addition, histone deacetylases (such as SlHDA1 and SlHDA3) and histone variants (such as Sl_H2A.Z) also play significant roles in the expression of related genes. Their expression or modification state changes are closely related to the content of lycopene (Ming et al., 2023). Members of the AP2 family of transcription factors (such as SlAP2c and SlAP2a) regulate histone acetylation levels and directly affect the expression of lycopene synthesis genes, thereby controlling lycopene accumulation in fruits (He et al., 2024). In addition, environmental stress, such as exposure to nanoplastics, can also indirectly affect lycopene content by altering DNA methylation and histone modification (Nazari et al., 2025). 6 Biotechnological and Breeding Applications 6.1 Epigenome editing tools: CRISPR/dCas9-based epigenetic regulation The CRISPR/dCas9 system can link inactivated Cas9 (dCas9) to epigenetic modification enzymes, such as DNA methyltransferases, demethylases or histone acetyltransferases. In this way, DNA methylation, demethylation or histone modification can be achieved at specific locations, thereby precisely controlling the expression of target genes (Figure 2) (Pan et al., 2021; Cai et al., 2023). For instance, dCas9 can bind to transcriptional activators (such as VP64, p300) or inhibitors (such as KRAB, DNMT3A), respectively activating the gene (CRISPRa) or silencing it (CRISPRi) This method can be used to regulate the key genes related to lycopene synthesis (Moradpour and Abdulah, 2019; Jogam et al., 2022). Furthermore, the CRISPR/dCas9 platform can simultaneously regulate multiple genes and even alter chromatin structure, providing a powerful tool for precisely controlling the lycopene metabolic pathway (Moradpour and Abdulah, 2019). 6.2 Marker-assisted and epigenetic breeding strategies for lycopene enhancement The combination of molecular marker-assisted selection (MAS) and epigenetic markers can accelerate the genetic improvement of traits related to lycopene content. Tiwari et al. (2023) found that through genome-wide association study (GWAS) and epigenomic sequencing, epigenetic variations closely related to lycopene accumulation could be identified, and then efficient molecular markers could be developed for breeding screening. Genome editing technologies such as CRISPR/Cas9 and CRISPR/dCas9 can directly act on the key genes in the lycopene synthesis pathway to achieve rapid and precise trait improvement (Chandrasekaran et al., 2021; Tan et al., 2024).
Molecular Plant Breeding 2025, Vol.16, No.5, 261-267 http://genbreedpublisher.com/index.php/mpb 265 Figure 2 Schematic of the CRISPR/dCas9 regulatory target gene (Adopted from Cai et al., 2023) Image caption: The expression vector expresses the dCas9 fusion protein in cells, which binds the transcribed sgRNA to form the CRISPR/dCas9 regulatory tool, resulting in the recruitment of the effector domain to the promoter or enhancer region of the target gene under the guidance of sgRNA. Effector domains act on promoters or enhancers of target genes to modify these regions, regulating target gene expression (Adopted from Cai et al., 2023) 6.3 Potential for integrating epigenomic data into tomato improvement programs With the development of high-throughput epigenomic sequencing technology, integrating multi-faceted epigenetic data such as DNA methylation, histone modification and non-coding RNA can more comprehensively analyze the regulatory network of lycopene accumulation (Moradpour and Abdulah, 2019). Combining epigenomic information with traditional genotype and phenotypic data can establish more accurate trait prediction models to assist in tomato variety improvement decisions (Karlson et al., 2021; Tiwari et al., 2023). In the future, the integration of epigenomic data is expected to promote molecular design breeding of lycopene high accumulation varieties and achieve targeted improvement of nutritional quality (Moradpour and Abdulah, 2019; Karlson et al., 2021). 7 Research Gaps and Future Perspectives 7.1 Limitations in current epigenomic studies on tomato carotenoids At present, there are still many deficiencies in the research on the epigenetic regulation of tomato carotenoids, especially lycopene. Although mechanisms such as DNA methylation, histone modification and non-coding RNA have been demonstrated to be related to fruit ripening and metabolic pathways, a complete regulatory network targeting key genes for lycopene synthesis has not been established, and systematic identification of related regulatory factors is also insufficient (Ming et al., 2023). Many studies only focus on a single epigenetic level and fail to delve into the interactions among different epigenetic modifications and their comprehensive regulatory effects on the entire carotenoid metabolic pathway. The mechanism by which environmental stress affects the epigenetic status and lycopene accumulation of tomatoes has not been systematically studied yet (Nazari et al., 2025).
Molecular Plant Breeding 2025, Vol.16, No.5, 261-267 http://genbreedpublisher.com/index.php/mpb 266 7.2 Multi-omics approaches for integrative understanding Multi-omics integration is an important future direction for solving these problems. The combination of data from genomics, epigenomics, transcriptomics, metabolomics, etc. can be used to analyze the dynamic relationship among epigenetic modifications, gene expression and metabolite accumulation. The histone variant Sl_H2A.Z has been proven to regulate the expression of genes related to carotenoid synthesis (Ming et al., 2023), but its synergistic effects with mechanisms such as DNA methylation and non-coding RNA still require multi-omics data for explanation. The influence of epigenetic changes caused by environmental factors on lycopene accumulation can be tracked and analyzed by multi-omics methods (Nazari et al., 2025). 7.3 Prospects for precision agriculture and metabolic engineering With the continuous revelation of epigenetic regulatory mechanisms, precision agriculture and metabolic engineering will have great potential in the targeted regulation of lycopene. By regulating key epigenetic factors, it is possible to achieve precise control of the lycopene synthesis pathway and improve the nutritional quality of fruits (Ming et al., 2023). Molecular breeding and gene editing technologies based on epigenetic markers will also provide new theoretical foundations and technical support for the breeding of varieties with high lycopene content. In the future, it is necessary to study the plasticity of epigenetic regulation under environmental stress to improve the stress adaptability and fruit quality of tomatoes (Nazari et al., 2025). Acknowledgments The authors thank Professor Gai for his meticulous guidance and valuable modification suggestions during the process of writing the manuscript of this study. Conflict of Interest Disclosure The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. References Alba R., Cordonnier-Pratt M., and Pratt L., 2000, Fruit-localized phytochromes regulate lycopene accumulation independently of ethylene production in tomato, Plant Physiology, 123(1): 363-370. https://doi.org/10.1104/PP.123.1.363 Apel W., and Bock R., 2009, Enhancement of carotenoid biosynthesis in transplastomic tomatoes by induced lycopene-to-provitamin a conversion, Plant Physiology, 151: 59-66. https://doi.org/10.1104/pp.109.140533 Cai R., Lv R., Shi X., Yang G., and Jin J., 2023, CRISPR/dCas9 tools: epigenetic mechanism and application in gene transcriptional regulation, International Journal of Molecular Sciences, 24(19): 14865. https://doi.org/10.3390/ijms241914865 Chandrasekaran M., Boopathi T., and Paramasivan M., 2021, A status-quo review on CRISPR-Cas9 gene editing applications in tomato, International Journal of Biological Macromolecules, 190: 120-129. https://doi.org/10.1016/j.ijbiomac.2021.08.169 Chen L., Meng J., He X., Zhang M., and Luan Y., 2018, Solanum lycopersicum microRNA1916 targets multiple target genes and negatively regulates the immune response in tomato, Plant, Cell and Environment, 42(4): 1393-1407. https://doi.org/10.1111/pce.13468 He X., Liu K., Wu Y., Xu W., Wang R., Pirrello J., Bouzayen M., Wu M., and Liu M., 2024, A transcriptional cascade mediated by two APETALA2 family members orchestrates carotenoid biosynthesis in tomato, Journal of Integrative Plant Biology, 66(6): 1227-1241. https://doi.org/10.1111/jipb.13650 Jogam P., Sandhya D., Alok A., Peddaboina V., Allini V., and Zhang B., 2022, A review on CRISPR/Cas-based epigenetic regulation in plants, International Journal of Biological Macromolecules, 219: 1261-1271. https://doi.org/10.1016/j.ijbiomac.2022.08.182 Karlson C., Mohd-Noor S., Nolte N., and Tan B., 2021, CRISPR/dCas9-based systems: mechanisms and applications in plant sciences, Plants, 10(10): 2055. https://doi.org/10.3390/plants10102055 Leiva-Ampuero A., Agurto M., Matus J., Hoppe G., Huidobro C., Inostroza-Blancheteau C., Reyes-Díaz M., Stange C., Canessa P., and Vega A., 2020, Salinity impairs photosynthetic capacity and enhances carotenoid-related gene expression and biosynthesis in tomato (Solanum lycopersicumL. cv. Micro-Tom), PeerJ, 8: e9742. https://doi.org/10.7717/peerj.9742 Liu L., Wei J., Zhang M., Zhang L., Li C., and Wang Q., 2012, Ethylene independent induction of lycopene biosynthesis in tomato fruits by jasmonates, Journal of Experimental Botany, 63: 5751-5761.
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Molecular Plant Breeding 2025, Vol.16, No.5, 268-277 http://genbreedpublisher.com/index.php/mpb 268 Feature Review Open Access Haplotype-Based Breeding of Yield-Related Traits in Oryza sativa: From Genomic Insights to Field Applications Qifu Zhang, Ruchun Chen, Zhongxian Li, Jianquan Li Hier Rice Research Center, Hainan Institute of Tropical Agricultural Resources, Sanya, 572025, Hainan, China Corresponding email: jianquan.li@hitar.org Molecular Plant Breeding, 2025, Vol.16, No.5 doi: 10.5376/mpb.2025.16.0027 Received: 11 Aug., 2025 Accepted: 13 Sep., 2025 Published: 20 Sep., 2025 Copyright © 2025 Zhang 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: Zhang Q.F., Chen R.C., Li Z.X., and Li J.Q., 2025, Haplotype-based breeding of yield-related traits in Oryza sativa: from genomic insights to field applications, Molecular Plant Breeding, 16(5): 268-277 (doi: 10.5376/mpb.2025.16.0027) Abstract This study summarizes the progress made in using haplotype analysis to investigate rice yield, reviews the role of methods such as QTL mapping, GWAS, and pan-genome in discovering genes related to yield, introduces how to identify functional haplotypes, and discusses how to prioritize them during selection. It also elaborates on several applications of haplotypes in breeding. For instance, marker-assisted selection, genomic selection, gene editing, etc. were discussed. The potential of combining multi-omics data, using artificial intelligence as predictive models, and considering the haplotypes of climate change in future molecular breeding was also explored. This study hopes to provide some theoretical and technical references for high-yield, stable-yield and climate change-adaptive rice breeding. Keywords Rice (Oryza sativa); Haplotype-based breeding; Yield-related traits; Molecular breeding; Multi-omics integration 1 Introduction The core of haplotype breeding is to identify and utilize good allele combinations. This can integrate the key genes related to yield together, improving the efficiency of breeding. Studies have found that haplotype combinations of different genotypes, especially functional haplotypes of core regulatory genes such as Ghd7, DTH8, and PRR37, can significantly improve traits such as grain count per panicle and 1000-grain weight of rice, thereby achieving high and stable yields (Sun et al., 2023; Sachdeva et al., 2024). In addition, the introduction of haplotypes from genetic resources such as wild rice also provides more materials for broadening the genetic basis of cultivated rice and discovering new superior haplotypes (Bharamappanavara et al., 2023). Nowadays, technologies such as high-throughput sequencing, genome-wide association study (GWAS), and molecular marker-assisted selection (MAS) are developing rapidly. This enables us to have a more systematic understanding of the genetic basis of rice yield traits. Many studies have identified a large number of haplotypes, quantitative trait loci (QTLs), and candidate genes related to yield and its components through GWAS and QTL mapping, and have also revealed their roles in different genetic backgrounds and environments (Ashfaq et al., 2023). Based on this, haplotype molecular design breeding and QTL pyramiding strategies have successfully achieved simultaneous improvement of multiple traits in the field, and new high-yield lines have also been cultivated (Withanawasam et al., 2022; Yadavalli et al., 2022). This study summarizes the latest progress in haplotype breeding of rice yield traits, introduces the application of haplotype analysis and genomic tools in yield improvement, explores how to identify excellent haplotypes, conduct functional analysis, and their practical application in molecular design breeding. By integrating genomic information and field performance, it promotes a better combination of theory and practice in high-yield rice breeding. This study aims to provide a theoretical basis and technical support for the precise improvement of rice yield traits in the future. 2 Genomic Basis of Yield-Related Traits in Rice 2.1 Key yield traits: grain size, grain number, panicle architecture, and biomass The yield of rice is determined by many complex traits together. It mainly includes grain size (grain length, grain width, grain thickness), the number of grains per panicle, the structure of the panicle (the length of the main
Molecular Plant Breeding 2025, Vol.16, No.5, 268-277 http://genbreedpublisher.com/index.php/mpb 269 panicle, the number of primary and secondary branches), and the biomass of the entire plant (Wang, 2024). These traits not only directly affect the yield, but also have a significant relationship with indirect traits such as photosynthetic efficiency, nutrient utilization, and stress resistance. Research has found that grain size, 1000-grain weight, number of grains per panicle, panicle length, and effective tillering number are the core traits that determine yield. There are obvious genetic correlations and phenotypic interactions among these traits (Ata-Ul-Karim et al., 2022; Yin et al., 2024). 2.2 Discoveries from QTL mapping, GWAS, and pan-genome studies Over the past three decades, researchers have identified many yield-related QTLs and candidate genes across the entire rice genome by using QTL mapping in parent populations and GWAS in natural populations. QTL mapping has identified major and minor loci that control traits such as grain weight, grain width, panicle length, and tillering number. Some QTLs exhibit stability in different genetic backgrounds and environments (Padmashree et al., 2023; Daryani et al., 2024). GWAS has made localization more precise and has also discovered many new pleiotropic loci and candidate genes. Some genes have a synergistic regulatory effect among multiple traits, such as GS3, GL3.1, OsCIPK17, GNP12, etc. (Yu et al., 2022; Roy et al., 2024). Meanwhile, pan-genome and large population sequencing studies have found that rice yield traits have strong polygenicity and complex genetic interaction networks. Major genes such as OsMADS22 and OsFTL1 have been functionally verified (Wang et al., 2020; Wei et al., 2024). 2.3 Functional validation of major yield-related genes Through gene cloning, expression analysis and mutant studies, many major yield genes have been functionally verified. For example, genes such as GS3, GW2, and GL3.1 respectively control grain length, grain width, and grain type; genes such as NOG1 and qHI6 affect sourge-reservoir relationship and grain filling; genes such as OsMADS22 and OsFTL1 regulate heading number and heading time (Khahani et al., 2020; Li et al., 2022; Wei et al., 2024). Furthermore, some QTLs and candidate genes have been applied in high-yield rice breeding through molecular marker-assisted selection (MAS) and gene editing techniques, significantly improving the efficiency of yield trait improvement (Kulkarni et al., 2020; Zhong et al., 2021). 3 Concept and Identification of Haplotypes 3.1 Definition of haplotypes and haplotype blocks in rice genomics Haplotype refers to a group of alleles or variant sites that are closely linked and inherited together on the same chromosome. In rice genomics, haplotype block usually refers to a region with a relatively high linkage disequilibrium (LD) in the population. These regions have less recombination and relatively stable allele combinations. The structure of haplotype blocks is related to factors such as ancestral recombination, natural selection, and population history, and is an important unit for studying the genetic basis of complex traits and conducting molecular breeding (Figure 1) (Zhang et al., 2021; Shipilina et al., 2022). Figure 1 Haplotype blocks defined through identity by descent (IBD) (Adopted from Shipilina et al., 2022)
Molecular Plant Breeding 2025, Vol.16, No.5, 268-277 http://genbreedpublisher.com/index.php/mpb 270 3.2 Haplotype detection tools: sequencing platforms, variant calling, and haplotype phasing The detection of haplotypes mainly relies on high-throughput sequencing platforms, such as Illumina short-read sequencing, Oxford Nanopore and PacBio long-read sequencing. By combining variant calling and haplotype phasing algorithms, haplotype information can be obtained. Commonly used phase determination tools include EAGLE2, BEAGLE, SHAPEIT2, etc. Different tools perform differently under different data types and group structures. There are mainly two methods for dividing haplotype blocks: the method based on linkage disbalance (LD) and the sliding window method. The LD method is usually more accurate. Analyzing with multiple tools together can make the phase determination results more reliable. In addition, new methods such as HaploBlocker can adapt to data with different marker densities and genetic diversity by focusing on the linkage structure within the population (Otte and Schlotterer, 2020; Weber et al., 2023). 3.3 Characterizing haplotype diversity across global rice germplasm collections Large-scale sequencing projects such as 3K-RG and MiniCore have revealed the wide distribution of haplotypes in the rice genome and their specificity among different subspecies and regions. Most haplotypes are from specific subspecies or specific populations. In the modern breeding process, the frequency of some major haplotypes has undergone significant changes. There are also many unique haplotypes between wild rice and cultivated rice, some of which have been selected during domestication and adaptation. In-depth research on haplotype diversity provides a solid foundation for the exploration of superior alleles and molecular design breeding (Shang et al., 2022; Aung et al., 2024; Huang et al., 2024). 4 Haplotype-Trait Associations 4.1 Statistical models linking haplotypes to yield phenotypes Methods such as genome-wide association study (GWAS) and mixed linear models (MLM) can control population structure and kinship, reducing false positives. GWAS, combined with the best linear unbiased prediction (BLUP) values and multi-year phenotypic data, can effectively identify haplotype blocks and candidate genes related to yield. Some researchers used 2.8 million SNPS and BLUP values to detect 816 SNP signals significantly related to 13 agronomic traits in 259 rice materials, and identified candidate genes through haplotype block construction (Wang et al., 2020; Wang et al., 2021; Wang et al., 2023). Anandan et al. (2022) and Al-Daej et al. (2023) found that mixed linear models and unified mixed models were also used for association analysis, reducing the interference caused by group structure and kinship. 4.2 Multi-environment validation of haplotype effects Verifying the results in different environments is an important step to ensure the reliability of haplotype-trait associations. Studies have shown that some haplotypes or QTLs can significantly affect yield traits in different ecological environments, different years and different genetic backgrounds. For instance, GWAS was conducted on traits such as flowering period under field and greenhouse conditions in the United States, Bangladesh and the United Kingdom, and it was found that ten genomic regions were associated with candidate genes in one or more environments. A single SNP can explain 5% to 50% of phenotypic variations. Some QTLs have stable effects in different geographical environments, indicating that these haplotypes have strong environmental adaptability and breeding potential (Bharamappanavara et al., 2023). 4.3 Functional haplotypes vs. neutral haplotypes: prioritization for breeding Functional haplotypes refer to haplotypes that have a significant impact on the target trait. Such haplotypes are preferred in molecular design breeding. Studies have found that in modern rice varieties, the frequencies of favorable haplotypes of most known yield-related genes are relatively low, indicating that by mining and aggregating these favorable haplotypes, it is expected to significantly increase the yield (Zhang et al., 2021; Wang et al., 2023). The prioritization of functional haplotypes usually takes into account their interpretability for phenotypes, stability across environments, and association with known functional genes. For instance, some haplotypes have been precisely localized in near-isogenic lines, showing significant structural and expression differences, which directly affect the expression of yield QTLs. On the contrary, neutral haplotypes have no significant impact on traits, so they are not given priority in breeding. The identification and sequencing of functional haplotypes provide a theoretical and practical basis for high-yield molecular breeding of rice.
Molecular Plant Breeding 2025, Vol.16, No.5, 268-277 http://genbreedpublisher.com/index.php/mpb 271 5 Breeding Strategies Using Superior Haplotypes 5.1 Marker-assisted haplotype selection and pyramiding Marker-assisted selection precisely screens and aggregates haplotypes related to the target trait by developing molecular markers that can distinguish different haplotypes. For important traits such as yield and stress resistance, haplotype analysis can identify “superior haplotypes” that are significantly superior to other haplotypes, and they can be aggregated into breeding materials through labeling assistance. Researchers have developed a series of haplotype-specific markers on traits such as grain weight and grain length in rice, which can effectively distinguish and track the transmission of superior haplotypes, improving genetic gain and breeding efficiency (Liu et al., 2023; Alam et al., 2024). Haplotype aggregation strategies can also help overcome linkage arrest by combining superior haplotypes of multiple genes, supporting the realization of “design breeding” (Sivabharathi et al., 2024; Meena et al., 2025). 5.2 Integration with genomic selection, speed breeding, and genome editing The combination of haplotype information and modern breeding techniques such as genome selection (GS), speed breeding, and genome editing can accelerate the utilization of superior haplotypes. Haplotype-based genomic selection models are more accurate than traditional SNP models in predicting complex traits such as yield and stress resistance, and can better reflect the complex relationship between genotype and phenotype (Figure 2) (Bhat et al., 2021; Yoosefzadeh-Najafabadi et al., 2022; Weber et al., 2023). Rapid incubation technology shortens the generation cycle, enabling superior haplotypes to aggregate and fix more quickly. Genome editing technology can also directly modify target genes, create or introduce superior haplotypes, and accelerate the creation of new varieties (Sivabharathi et al., 2024; Meena et al., 2025). Figure 1 Mining of SNPs and construction of haplotypes for detecting marker-trait associations (GWAS) and computing genomic estimated breeding values (GS) (Adopted from Bhat et al., 2021) Image caption: This diagram describes the comparative potential of the Haplotype-Based GWAS/Haplotype-Based GS in relation to SNP-Based GWAS/SNP-Based GS for the development of improved crop cultivars via genomics-assisted breeding (GAB). It showed that Haplotype-Based GWAS/Haplotype-Based GS in combination with the high-throughput phenotyping (HTP) has great potential to enhance the precision and accuracy in the gene identification and GAB (Adopted from Bhat et al., 2021) 5.3 Combining haplotype information with hybrid rice breeding programs Haplotype information has brought new ideas for parent selection and hybrid combination design in hybrid rice breeding. Liu et al. (2023) and Singh et al. (2024) found that by analyzing the haplotypes of key genes in parental materials such as restorer lines and maintainer lines, parents carrying superior haplotypes can be selected, thereby enhancing the yield potential and stability of hybrid offspring. The utilization of haplotype diversity can also broaden the genetic basis of hybrid rice and enhance its stress resistance and adaptability. Studies have shown that
Molecular Plant Breeding 2025, Vol.16, No.5, 268-277 http://genbreedpublisher.com/index.php/mpb 272 the combination of superior haplotype aggregation and hybrid rice breeding can simultaneously improve multiple traits such as yield and disease resistance, providing a reliable genomic basis for the continuous innovation of hybrid rice (Sinha et al., 2020). 6 Case Study: Haplotype-Based Improvement of Yield Traits 6.1 Choice of target yield traits and rice populations Traits such as grain weight, the number of grains per panicle, panicle length, plant height, flag leaf size and biomass are the main improvement targets of rice haplotype breeding. These traits not only directly affect the yield, but are also closely related to stress resistance and adaptability. Studies usually employ populations with diverse genetic backgrounds, including cultivated rice (indica rice, japonica rice), wild rice (O. rufipogon, O. glaberrima), and their backcross derivative lines. This can maximize the exploration and utilization of beneficial haplotypes (Ashfaq et al., 2023; Bharamappanavara et al., 2023; Udaya et al., 2023). For instance, some studies analyzed the effects of GRF4 haplotypes on yield and biomass using 335 rice samples, or systematically evaluated the contributions of different haplotypes to yield traits by constructing an introduction line library containing multiple AA genomic germplasm (Zhang et al., 2022; Sahoo et al., 2024). 6.2 Experimental workflow: sequencing, haplotype analysis, and candidate selection The experimental process generally includes high-throughput genomic sequencing, SNP typing, haplotype construction, association study (GWAS or QTL mapping), and candidate haplotype screening. Firstly, conduct genome-wide SNP testing on the target population and perform association analysis in combination with phenotypic data to identify haplotypes or QTLS that are significantly associated with yield traits. For instance, GWAS could detect multiple major and multi-effect loci related to traits such as grain weight, panicle length, and seed setting rate in 100 to 400 diverse materials (Ashfaq et al., 2023). Then, through molecular marker-assisted selection (MAS) or gene editing techniques (such as CRISPR/Cas9), superior haplotypes were introduced into breeding materials to achieve precise improvement (Sahoo et al., 2024). 6.3 Field performance, yield gains, and scalability of breeding outcomes Haplotype improved materials have demonstrated significant yield increases and trait stability in multi-environment field trials. The superior haplotype of GRF4 (Hap1) can increase yield and biomass. Some QTL polymer lines have yields more than 50% higher than control varieties under drought or high-temperature stress (Withanawasam et al., 2022; Zhang et al., 2022; Sahoo et al., 2024). The introduced lines obtained by introducing the superior haplotypes of wild rice into the cultivated rice background showed high yield and wide adaptability in different genetic backgrounds and ecological regions (Zhang et al., 2022; Bharamappanavara et al., 2023). Haplotype breeding strategies can specifically enhance yield traits and have excellent scalability and application prospects. 7 From Genomic Insights to Field Applications 7.1 Multi-location trials for stability and environmental adaptability Field trials of rice materials at multiple locations and in different seasons can effectively identify haplotype combinations that are high-yielding and stable in various environments. James et al. (2024) utilized backcrossover introduction systems and, through three consecutive seasons of field trials, combined with statistical methods such as AMMI and GGE, screened out materials that performed excellently in various environments, providing a scientific basis for subsequent large-scale promotion. Evaluating the environmental adaptability of superior haplotypes is helpful for identifying genotypes with broad adaptability or specific adaptability and enhancing the application value of new varieties under complex ecological conditions (Bharamappanavara et al., 2023). 7.2 Integration with precision agriculture and digital phenotyping tools Tools such as high-throughput phenotypic platforms, remote sensing technologies and big data analysis can monitor the growth and yield traits of rice in the field in real time and non-destructive, accelerating the screening and evaluation of superior haplotypes (Bhat et al., 2021). Predictive models that combine genomic selection (GS) and machine learning have enhanced the prediction accuracy and breeding decision-making efficiency of complex yield traits by leveraging large-scale phenotypic and genotypic data (Bejjam and Basuthkar, 2024). The
Molecular Plant Breeding 2025, Vol.16, No.5, 268-277 http://genbreedpublisher.com/index.php/mpb 273 combination of these technologies enables genomic information to be transformed into field performance more quickly, providing strong technical support for high-yield rice breeding (Bhat et al., 2021). 7.3 Farmer participatory breeding and real-world adoption challenges Farmer participatory breeding can make haplotype breeding more closely integrated with actual production demands. Allowing farmers to directly participate in variety selection and field trials can better identify excellent haplotype materials that not only meet agronomic requirements but also are suitable for the local environment. However, there are still many challenges in the actual promotion, such as farmers’ acceptance of new varieties, the matching of variety quality and market demand, technical services and policy support during the promotion process, etc. Although some farmers' selected strains have high yield and early maturity, they have deficiencies in terms of quality and other aspects, and ultimately failed to be widely promoted. This also reminds us that haplotype breeding must take into account multiple traits and market demands in practical applications (James et al., 2024). 8 Challenges and Research Gaps 8.1 Haplotype resolution limits in complex genomes The yield traits of rice are controlled by many genes, and there are also complex interactions and linkage disequilibrium among these genes. This makes haplotype analysis face the problem of insufficient resolution in the context of complex genomes. Although high-throughput sequencing and GWAS technologies have facilitated the localization of QTLS and candidate genes, polyalleles, gene interactions, and environmental influences have made the precise analysis and functional verification of haplotypes very difficult. Especially in the context of the introduction of intersubspecies or wild rice, genomic structural variations and segregation biases complicate the problem (Adam et al., 2023; Bharamappanavara et al., 2023; Sachdeva et al., 2024). 8.2 Data integration: linking genomics, transcriptomics, and phenomics At present, the integrated analysis of genomic, transcriptomic and phenome data is still insufficient, which hinders the in-depth understanding of the regulatory network of complex traits. Multi-omics joint analysis has been initially applied in candidate gene mining and functional annotation, but the efficiency of data standardization, heterogeneity processing and large-scale data integration remains a technical challenge. The significant variations in the field environment and the insufficient popularity of high-throughput phenotypic techniques have also affected the accuracy of genotype-phenotypic associations (Kiranmai, 2023; Bejjam and Basuthkar, 2024; Sachdeva et al., 2024). 8.3 Socio-economic and policy considerations for large-scale deployment In the field application and large-scale promotion of haplotype breeding, it is not only necessary to solve technical problems, but also to take into account multiple factors such as society, economy and policy. The promotion of superior haplotype varieties is restricted by farmers’ acceptance of new varieties, the construction of seed systems, intellectual property protection and policy support, etc. Especially in developing countries, insufficient resource allocation, inadequate technical training and weak infrastructure have all affected the implementation of haplotype breeding achievements. Moreover, policies on biodiversity conservation and sustainable agriculture have also put forward higher requirements for the promotion of new varieties (Demeke et al., 2022; Withanawasam et al., 2022; Pallavi et al., 2024). 9 Future Prospects 9.1 Multi-omics-driven haplotype discovery for complex traits By integrating multi-omics data, researchers can more comprehensively identify key genes and superior haplotypes related to complex traits such as yield and stress resistance. The combination of genome-wide association study (GWAS) and multi-omics platforms can reveal functional genes and their regulatory networks that regulate traits such as yield and drought resistance, providing theoretical basis and molecular markers for precision breeding (Mahmood et al., 2022). The combination of phenotype, genotype and multi-omics data can significantly improve the prediction accuracy of complex traits such as hybrid rice (Xu et al., 2020; Hu et al., 2021). In the future, with the rapid accumulation and analysis of multi-omics data, the discovery and application
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