Uncovering the genetic architecture of pungency, carotenoids, and flavor in Capsicum chinense via TWAS-mGWAS integration and spatial transcriptomics

Abstract

Capsicum chinense (habanero pepper) exhibits substantial variation in fruit pungency, color, and flavor due to its rich secondary metabolite composition, including capsaicinoids, carotenoids, and volatile organic compounds (VOCs). To dissect the genetic and regulatory basis of these traits, we conducted an integrative analysis across 244 diverse accessions using metabolite profiling, genome-wide association studies (GWAS), and transcriptome-wide association studies (TWAS). GWAS identified 507 SNPs for capsaicinoids, 304 for carotenoids, and 1176 for VOCs, while TWAS linked gene expression to metabolite levels, highlighting biosynthetic and regulatory genes in phenylpropanoid, fatty acid, and terpenoid pathways. Segmental RNA sequencing across fruit tissues of contrasting accessions revealed 7034 differentially expressed genes, including MYB31, 3-ketoacyl-CoA synthase, phytoene synthase, and ABC transporters. Notably, AP2 transcription factors and Pentatrichopeptide repeat (PPR) emerged as central regulators, co-expressed with carotenoid and VOC biosynthetic genes. High-resolution spatial transcriptomics (Stereo-seq) identified 74 genes with tissue-specific expression that overlap with GWAS and TWAS loci, reinforcing their regulatory relevance. To validate these candidates, we employed CRISPR/Cas9 to knock out AP2 and PPR genes in tomato. Widely targeted metabolomics and carotenoid profiling revealed major metabolic shifts: AP2 mutants accumulated higher levels of β-carotene and lycopene. In contrast, PPR mutants altered xanthophyll ester and apocarotenoid levels, supporting their roles in carotenoid flux and remodeling. This study provides the first integrative GWAS-TWAS-spatial transcriptomics in C. chinense, revealing key regulators of fruit quality traits. These findings lay the groundwork for precision breeding and metabolic engineering to enhance nutritional and sensory attributes in peppers.

Acknowledgments

The USDA-ARS Germplasm Resources Information Network (GRIN) for the Capsicum chinense accessions. The authors are thankful to Ms. Lakshmi Abburi and Suresh Alaparthi for technical help throughout the investigation. We acknowledge Manohar Chakrabarthi for the valuable suggestions. This study was supported by the National Institute of Food and Agriculture USDA-NIFA (grant no. 2018-04969, 2021-12930, 2022-10141, and wvax-EA-Padma-2024) and the National Science Foundation (NSF Award Number 2318707).

Author contributions

All authors have read and agreed to the published version of the manuscript. Padma Nimmakayala, Umesh K. Reddy (Conceptualization), Padma Nimmakayala (Data curation), Padma Nimmakayala, Krishna Sai Karnatam (Formal analysis), Padma Nimmakayala, Donald A. Adjeroh, Umesh K. Reddy (Funding acquisition), Umesh K. Reddy, Padma Nimmakayala, Krishna Sai Karnatam, Alicia Talavera-Caro, Kang-Mo Ku, Sai Satish Kadiyala, Prapooja Somagattu, Ritik Duhan, Sahithi Ramireddy, Subramanyam Reddy Chinreddy, Carlos Lopez-Ortiz (Investigation), Umesh K. Reddy, Krishna Sai Karnatam, Kang-Mo Ku, Purushothaman Natarajan, Alicia Talavera-Caro, Sai Satish Kadiyala, Sahithi Ramireddy, Subramanyam Reddy Chinreddy (Methodology), Padma Nimmakayala, Umesh K. Reddy (Project administration), Umesh K. Reddy, Alicia Talavera-Caro, Purushothaman Natarajan, Sai Satish Kadiyala, Virender Kumar, Donald A. Adjeroh (Software), Padma Nimmakayala, Umesh K. Reddy (Supervision), Alicia Talavera-Caro (Validation), Padma Nimmakayala, Umesh K. Reddy (Visualization), Umesh K. Reddy, Krishna Sai Karnatam, Alicia Talavera-Caro, Carlos Lopez-Ortiz, Padma Nimmakayala, Donald A. Adjeroh, Vagner A. Benedito (Writing—original draft), and Umesh K. Reddy, Carlos Lopez-Ortiz, Krishna Sai Karnatam, Alicia Talavera-Caro, Padma Nimmakayala, Donald A. Adjeroh, Vagner A. Benedito (Writing—review & editing).

Data availability

The genotyping by sequencing data underlying this article are available in the NCBI database at https://www.ncbi.nlm.nih.gov/sraand can be accessed with the BioProject accession number PRJNA1305095. The raw paired-end of the Segmental RNA-Seq data generated during this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1130500.

Conflict of interest statement

The authors declare no conflict of interest.

Supplementary Data

Supplementary data is available at Horticulture Research online.

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