A Genome-Wide Association Study Reveals QTLs and Candidate Genes Associated with the Carotenoid Content in the Flesh of Cucurbita pepo L. Fruit Alba López, Alicia García, Alejandro Castro-Cegrí, María Segura, Álvaro Benítez, Francisco Palma, Dolores Garrido, Cecilia Martínez, Manuel Jamilena Antioxidants, 2025 Considering the importance of carotenoids in the human diet, their enhancement is a key trait in current breeding programs. This study assessed lutein, zeaxanthin, α-carotene, and β-carotene levels in the flesh of mature fruits from 257 global C. pepo accessions. Lutein and β-carotene were the most prevalent, with top accessions identified for each carotenoid. A panel of 120 accessions with reliable carotenoid contents and genetic diversity was analyzed using 23,111 GBS-generated SNPs in genome-wide association studies (GWAS). Three genomic regions (qtl1, qtl3, and qtl13) on chromosomes 1, 3, and 13 were significantly linked to carotenoid levels, with alternative alleles increasing the carotenoid content, leading to yellowish–orange flesh. Seven candidate genes were identified: CpTIC56, CpHSHP70, and CpPDL8, which regulate carotenoid biosynthesis in chloroplasts; CpSPX and CpPHO1, associated with phosphate homeostasis and carotenoid buildup; CpMYB106, co-expressed with carotenoid biosynthesis genes; and a CpPPR RNA-binding protein. RNA-seq data from yellow- and white-fleshed fruits supported their involvement in carotenoid accumulation. These results improve our understanding of the genetic control of carotenoid buildup in C. pepo fruit, supporting breeding efforts for improved nutritional quality.
RNA-Seq-based analysis of transcriptomic signatures elicited by mutations conferring salt tolerance in Cucurbita pepo Keshav Gautam, Sonsoles Alonso, Alicia García, María Segura, Álvaro Benítez, Cecilia Martínez, Manuel Jamilena Plant Stress, 2025 • Pairwise comparisons of transcriptomes of wild-type and salt-tolerant mutants reveal the molecular mechanisms behind salt tolerance in squash. • Brassinosteroid, ABA, ethylene, and JA biosynthesis genes like CpBZR1, CpTCH4–1, CpTCH4–2, CpCYP707A1, CpACO1–2 , and CpLOX3 were key regulators of salt tolerance in Cucurbita . • Salt-tolerance-associated DEGs were significantly enriched in plant hormone, MAPK signalling, carotenoid biosynthesis and starch/sucrose metabolism pathways. • Squash response to salt stress involves regulation of several transcription factors of the ERF, C3H, MYB, HSF, NAC, knotted and WRKY families, along with several Lncr loci. Salinity is a major determinant of plant growth and crop productivity, resulting in significant economic losses in agriculture. Improving salinity tolerance in plant breeding programs requires not only donor tolerant genotypes but also a thorough knowledge of the genes controlling the trait. Taking advantage of two recently identified salinity-tolerant EMS mutants of squash ( sal-1 and sal-2 ), this study aimed to analyse whether these two sources of salt tolerance are associated with similar transcriptomic changes in leaves. RNA sequencing revealed that the two mutants have a very distinct transcriptomic response to salt stress compared to the WT, with 154 and 1068 salt-tolerance-associated differentially expressed genes (DEGs) in sal-1 and sal-2 , respectively. GO and KEGG enrichment analyses revealed the importance of several phytohormone biosynthesis, signalling and transport genes ( CpAUX22B/22D, CpSAUR32–2, CpARR5/12, CpAHK2/3, CpBZR1, CpTCH4, CpNCED1, CpCYP707A1, CpPP2C, CpSnRK1/2, CpLOX2 and CpACX ) in the salt tolerance response. MAPK genes ( CpMPK3 and CpMEKK1 ) and the Ca²⁺ signalling network ( CpCPK26/28/34, CpCML31/36/48, CpPBP1, CpCBL1 and CpRBOHD ) were also specifically activated in salt-tolerant mutants, indicating their contribution to salt tolerance. Genes for antioxidant enzymes (PP2, POD, CAT, PRX, GST and GRX) and cell wall metabolism were also up-regulated in salt-tolerant mutants, reducing oxidative stress and maintaining the integrity of membranes and other cellular structures. Genes for ion transporters were significantly up-regulated in response to salt stress in sal-2 , probably involved in maintaining ion homeostasis. Several genes encoding transcription factors of the ERF, C3H, Dof, HD-ZIP, MYB, HSF, NAC, knotted and WRKY families, as well as long non-coding RNA, were also found to positively or negatively regulate salt stress tolerance in the sal-1 and sal-2 mutants. Overall, the results highlight the complexity of the molecular response involved in salt stress tolerance in C. pepo and prioritise further investigation of specific genes that contribute to the resilience of crops under saline conditions.
High throughput screening and evaluation of salt-tolerant mutants from an EMS collection of Cucurbita pepo Sonsoles Alonso, Keshav Gautam, Jessica Iglesias-Moya, María Segura, María del Mar Rebolloso, María del Carmen Salas-Sanjuán, Cecilia Martínez, Manuel Jamilena Frontiers in Plant Science, 2025 Salinity is a major environmental stress limiting crop growth and yield in many arid and semi-arid regions of the world. In this study, we used a germination-based selection method to screen an EMS collection of Cucurbita pepo, consisting of 3,751 M2 lines, for salt tolerance. The screening resulted in the identification of six salt-tolerant mutants that exhibited enhanced germination and plant growth under salt stress conditions. This enhanced salt tolerance was found to be associated with increased production of proline, soluble sugars, and anthocyanins, which are known to exert osmoprotective and ROS scavenging functions. The mutant lines TS-1378 and TS-2075 were selected to be tested as rootstocks and showed a positive effect on the vegetative growth of scions under standard and saline conditions. In conclusion, the research provides an efficient protocol for high throughput screening for salt tolerance, novel useful mutants to study the mechanism behind salt tolerance, and valuable genetic resources for pumpkin breeding.
Structural and functional characterization of genes PYL-PP2C-SnRK2s in the ABA signalling pathway of Cucurbita pepo Jessica Iglesias-Moya, Álvaro Benítez, María Segura, Sonsoles Alonso, Dolores Garrido, Cecilia Martínez, Manuel Jamilena BMC Genomics, 2024 BackgroundThe core regulation of the abscisic acid (ABA) signalling pathway comprises the multigenic familiesPYL,PP2C,andSnRK2. In this work, we conducted a genome-wide study of the components of these families inCucurbita pepo.ResultsThe bioinformatic analysis of theC. pepogenome resulted in the identification of 19CpPYL, 102CpPP2Cand 10CpSnRK2genes. The investigation of gene structure and protein motifs allowed to define 4 PYL, 13 PP2C and 3 SnRK2 subfamilies. RNA-seq analysis was used to determine the expression of these gene families in different plant organs, as well as to detect their differential gene expression during germination, and in response to ABA and cold stress in leaves. The specific tissue expression of some gene members indicated the relevant role of some ABA signalling genes in plant development. Moreover, their differential expression under ABA treatment or cold stress revealed those ABA signalling genes that responded to ABA, and those that were up- or down-regulated in response to cold stress. A reduced number of genes responded to both treatments. SpecificPYL-PP2C-SnRK2genes that had potential roles in germination were also detected, including those regulated early during the imbibition phase, those regulated later during the embryo extension and radicle emergence phase, and those induced or repressed during the whole germination process.ConclusionsThe outcomes of this research open new research lines for agriculture and for assessing gene function in future studies.
A QTL on chromosome 17 identified by Genome-Wide Association Mapping controls postharvest cold tolerance of Cucurbita pepo L. Alicia García, Alejandro Castro‐Cegrí, Alba López, María Segura, Álvaro Benítez, Dolores Garrido, Francisco Palma, Cecilia Martínez, Manuel Jamilena Physiologia Plantarum, 2024 The worldwide cultivated Cucurbita pepo L. is one of the most diverse species in the plant kingdom. In this study, chilling tolerance over a wide range of cultivars was characterized to discover the allelic variants to improving the postharvest quality of the immature fruit during cold storage. For this purpose, fruits from 126 accessions of worldwide origin have been evaluated for weight loss and chilling injury after 3, 7 and 14 days of cold storage, classifying them into tolerant, partially tolerant, and sensitive accessions. To verify this classification, antioxidant capacity and lipid peroxidation (MDA) of contrasting accessions (tolerant vs. sensitive) were assessed. The antioxidant capacity significantly decreased during cold storage in the sensitive accessions, while it was maintained in tolerant accessions. Additionally, the sensitive accessions presented a higher accumulation of MDA during this period. Finally, a GWAS analysis using GBS data available in CuGenDBv2, combined with weight loss percentage data, led to the identification of a candidate QTL located on chromosome 17 that regulates postharvest cold tolerance in zucchini. The region contains four SNPs whose alternative alleles were significantly associated with lower weight loss percentage and chilling injury indices during cold storage. Two SNPs are located in the 3’ UTR region of the gene CpERS1, a gene involved in ethylene perception. The other two SNPs generate missense mutations in the coding region of a Pectin methyl esterase inhibitor gene (CpPMI). The role of this QTL and these variants in chilling tolerance is discussed.
Jasmonate-insensitive mutant jar1b prevents petal elongation and flower opening coupling with parthenocarpic fruit development in Cucurbita pepo Keshav Gautam, María Segura, Sonsoles Alonso, Raúl Pasadas, José M. García-Mina, Angel M. Zamarreño, Cecilia Martínez, Manuel Jamilena Plant Physiology and Biochemistry, 2024 Jasmonates are growth regulators that play a key role in flower development, fruit ripening, root growth, and plant defence. The study explores the coordination of floral organ maturation to ensure proper flower opening for pollination and fertilization. A new mutant (jar1b) was discovered, lacking petal elongation and flower opening but showing normal pistil and stamen development, leading to parthenocarpic fruit development. The mutation also enhanced the elongation of roots while reducing the formation of root hairs. BSA sequencing showed that jar1b is a missense mutation in the gene CpJAR1B, which encodes the enzyme that catalyzes the conjugation between JA and the amino acid isoleucine. The loss of function mutation in CpJAR1B produced a deficiency in biologically active (+) -7-iso-jasmonoyl-L-isoleucine (JA-Ile), which was not complemented by the paralogous gene CpJAR1A or any other redundant gene. Exogenous application of methyl jasmonate (MeJA) demonstrated that jar1b is partially insensitive to JA in both flowers and roots. Further experimentation involving the combination of JA-Ile deficient and ethylene-deficient, and ET insensitive mutations in double mutants revealed that CpJAR1B mediated ET action in female petal maturation and flower opening, but JA and ET have independent additive effects as negative regulators of the set and development of squash fruits. CpJAR1B also regulated the aperture of male flowers in an ethylene-independent manner. The root phenotype of jar1b and effects of external MeJA treatments indicated that CpJAR1B has a dual role in root development, inhibiting the elongation of primary and secondary roots, but promoting the formation of root hairs.
The transcription factor CpMYB62 controls the genetic network that leads to the determination of female flowers in Cucurbita pepo María Segura, Alicia García, German Gamarra, Álvaro Benítez, Jessica Iglesias-Moya, Cecilia Martínez, Manuel Jamilena Horticulture Research, 2024 In monoecious species, female flowering constitutes the developmental process that determines the onset and production of fruit and is therefore closely related to crop yield. This article presents the identification and phenotypic and molecular characterization of myb62, an ethylmethane sulfonate loss-of-function mutation that completely blocks the female floral transition, converting all female flowers into male flowers. BSA-seq analysis coupled with WGS showed that myb62 corresponds to a C>T transition in the coding region of the gene CpMYB62, generating a premature stop codon and a truncated transcription factor without its N-terminal effector domain. The myb62 phenotype was partially rescued by exogenous ethylene application, indicating that the function of CpMYB62 is mediated by ethylene. Different evidence supports this conclusion: first, the reduced ethylene production of the mutant, and second, the male flower productive phenotype of the double mutant between myb62 and the ethylene-insensitive mutant etr2b, which demonstrated that myb62 is epistatic over etr2b. Furthermore, transcriptomic analysis of WT and myb62 apical shoots confirmed that CpMYB62 regulates master sex-determining genes, upregulating those encoding the ethylene biosynthesis enzymes CpACO2B and CpACS27A and those encoding for transcription factors that promote the development of carpels(CpCRC), but downregulating those involved in the arrest of carpels (CpWIP1), In the gene network controlling sex determination in cucurbits, CpMYB62 occupies the most upstream position, activating ethylene and other sex determining genes involved in female flower determination in Cucurbita pepo.
An miR164-resistant mutation in the transcription factor gene CpCUC2B enhances carpel arrest and ectopic boundary specification in Cucurbita pepo flower development María Segura, Alicia García, Germán Gamarra, Álvaro Benítez, Jessica Iglesias-Moya, Cecilia Martínez, Manuel Jamilena Journal of Experimental Botany, 2024 The sex determination process in cucurbits involves the control of stamen or carpel development during the specification of male or female flowers from a bisexual floral meristem, a function coordinated by ethylene. A gain-of-function mutation in the miR164-binding site of CpCUC2B, ortholog of the Arabidopsis transcription factor gene CUC2, not only produced ectopic floral meristems and organs, but also suppressed the development of carpels and promoted the development of stamens. The cuc2b mutation induced the transcription of CpCUC2B in the apical shoots of plants after female flowering but repressed other CUC genes regulated by miR164, suggesting a conserved functional redundancy of these genes in the development of squash flowers. The synergistic androecious phenotype of the double mutant between cuc2b and etr2b, an ethylene-insensitive mutation that enhances the production of male flowers, demonstrated that CpCUC2B arrests the development of carpels independently of ethylene and CpWIP1B. The transcriptional regulation of CpCUC1, CpCUC2, and ethylene genes in cuc2b and ethylene mutants also confirms this conclusion. However, the epistasis of cuc2b over aco1a, a mutation that suppresses stamen arrest in female flowers, and the down-regulation of CpACS27A in cuc2b female apical shoots, indicated that CpCUC2B promotes stamen development by suppressing the late ethylene production.
Comparative RNA-Seq Analysis between Monoecious and Androecious Plants Reveals Regulatory Mechanisms Controlling Female Flowering in Cucurbita pepo María Segura, Alicia García, Álvaro Benítez, Cecilia Martínez, Manuel Jamilena International Journal of Molecular Sciences, 2023 In the monoecious Cucurbita pepo, the transition to female flowering is the time at which the plant starts the production of female flowers after an initial male phase of development. Ethylene plays an essential role in this process since some ethylene deficient and ethylene-insensitive mutants are androecious and only produce male flowers. To gain insight into the molecular mechanisms regulating the specification and early development of female flowers, we have compared the transcriptomic changes occurring in the shoot apices of WT and androecious ethylene-insensitive etr1b mutant plants upon female flowering transition. There were 1160 female flowering-specific DEGs identified in WT plants upon female flowering, and 284 of them were found to be modulated by the ethylene-insensitive etr1b mutation. The function of these DEGs indicated that female flower specification depends on the adoption of a transcriptional program that includes previously identified sex-determining genes in the ethylene pathway, but also genes controlling the biosynthesis and signaling pathways of other phytohormones, and those encoding for many different transcription factors. The transcriptomic changes suggested that gibberellins play a negative role in female flowering, while ethylene, auxins, ABA and cytokinins are positive regulators. Transcription factors from 34 families, including NAC, ERF, bHLH, bZIP, MYB and C2H2/CH3, were found to be regulating female flowering in an ethylene-dependent or -independent manner. Our data open a new perspective of the molecular mechanisms that control the specification and development of female flowers in C. pepo.
