Recreating viable YY^h genotype uncovers the role of CpYYL underlying YY lethality in papaya

Domain: neuroscience
Published: 2026-01-24
Editorial outcome: major_revision

Abstract
Papaya is a trioecious species with XX females, XY males, and XYh hermaphrodites. Any combination of Y and Yh chromosomes is lethal. The gene underlying YY lethality is pivotal in the transition from stage 2 to stage 3 in sex chromosome evolution. Here, we identify and functionally validate the YY lethality gene in papaya, CpYYL. Loss of CpYYL function accelerates glycolysis and reduces sucrose accumulation in embryo, causing embryo abortion. CpYYL interacts with CpAKRP and overexpression of both genes partially rescues seed abortion in the heterozygous emb506 and akrp mutants, indicating conserved role in plastid differentiation and embryo development. Viable YYh and YhYh plants are generated through genetic engineering and crossing, although both genotypes show high seedling mortality. Crossing YYh male with XX female papaya shows 1:1 ratio of male to hermaphrodite plants without recombination between MSY and HSY. Identification of CpYYL fills a critical gap in papaya sex chromosome evolution, and provides new resources for genomic and epigenetic studies.

Introduction
Papaya (Carica papaya L., 2n=2x=18) is a fast-growing fruit crop known for its palatable flavor, rich nutritional profile, and versatile culinary and medicinal applications. It is native to Central America and southern Mexico and is now cultivated in tropical and subtropical regions worldwide. The papaya fruit is a large, fleshy berry that varies in size, shape, and color depending on the variety. Papaya pulp is rich in provitamin A, vitamin C, and other essential nutrients, making it a popular choice for fresh consumption and processed products like juices, jams, and dried fruit. The protease enzyme papain is widely used in the food, pharmaceutical, and cosmetic industries. Papaya has been recommended as a dietary intervention to prevent vitamin A deficiency in developing countries1.

Papaya is a diploid species with 9 chromosomes and a genome size of 352Mb2. Transgenic papaya has been widely publicized as a success story that saved the papaya industry from an epidemic of papaya ringspot virus (PRSV)3,4. Since the devastating PRSV disease has been effectively controlled, the primary problem for papaya production is the segregation of sex types. In most papaya-producing regions, hermaphrodites are preferred due to their higher productivity and compact, pear-shaped fruits, which reduce shipping costs by 20%. However, the progeny from self-fertilized hermaphrodites segregate in a 2:1 ratio (two hermaphrodites to one female), while progeny from female trees pollinated by hermaphrodites segregate in a 1:1 ratio (one hermaphrodite to one female). This occurs because YhYh embryos are nonviable. Farmers relying on hermaphrodites for production need to plant at least five seedlings per planting hill and wait 4–6 months until flowering. Thinning is then performed to maximize the number of hermaphrodite plants. This practice is labor-intensive and inefficient, resulting in wasted time, water, and nutrients while delaying production due to competition among the plants in early growth. This has been a problem since the beginning of papaya cultivation that persists to this day5. Identifying the gene responsible for YY lethality could pave the way for engineering viable YYh and YhYh genotypes. These genotypes are essential germplasms for developing true-breeding hermaphrodite cultivars by crossing XX females with YhYh hermaphrodites.

Dioecy and sex chromosomes are common in animals but rare in plants, occurring in approximately 10% land plant species and 6% of flowering plant species6,7. The evolution of sex chromosomes can be categorized into six stages. Stage 3 involves the expansion of the male-specific region due to the accumulation of retrotransposons, translocation and duplication of genomic fragments. This process is accompanied by Y chromosome degeneration, resulting in the lethality of the YY genotype. Consequently, males are restricted to the heterozygous XY genotype, as observed in papaya6.

Cultivated hermaphrodite papaya was selected from a male population in Central America about 4000 years ago, resulting in a modified Yh chromosome8. The male- and hermaphrodite-specific region of the Y and Yh chromosomes (MSY and HSY) has been sequenced9. The identity of MSY and HSY is 99.6%, indicating they share a common ancestral MSY recently8.

Extensively studied plant sex chromosomes include those of white campion (Silene latifolia Poir.)10–12, papaya8,9, and asparagus (Asparagus officinalis L.)13,14. The successful assembly of the asparagus YY genome led to the identification of two sex determination genes in the asparagus MSY14. A major challenge in studying MSY in papaya and white campion is the unavailability of YY genotype. In the XY genotype, the X chromosome does not recombine with MSY, rendering genes in MSY genetically unmappable15,16. This limitation could be overcome if viable YY genotypes of papaya could be recreated.

Classical genetic analyses led to the hypothesis of five factors in the sex determination region, contributing to distinctive morphologies among the three sex types in papaya17. 1. Mp, male peduncle, responsible for the long, ramified, many-flowered cymose inflorescence of male trees; 2. l, zygotic lethal (i.e., YY lethality); 3. sa, suppressor of the androecium; 4. sg, suppressor of the gynoecium; 5. C, hypothetical factor for recombination suppression in the sex determining region. We have discovered that the factor C is two inversions that occurred 7 and 1.9 million years ago (mya), respectively9. Together, these five factors—four genes and two chromosomal rearrangements—are essential for papaya sex chromosomes to reach stage 3, characterized by YY lethality. The objectives of this study are to identify the YY lethality gene and generate viable YhYh hermaphrodites for papaya improvement.

In this work, we identify CpYYL as the gene responsible for YY lethality in papaya, a defining feature of its evolved sex chromosomes. Using transgenic complementation, we generate viable YYh and YhYh plants, providing critical resources for dissecting sex chromosome recombination. We further demonstrate that CpYYL interacts with CpAKRP and that loss of CpYYL disrupts sucrose metabolism, leading to embryo abortion. These findings directly link the degeneration of an essential gene on the Y chromosome to the stabilization of dioecy, offering evolutionary insights and valuable germplasm for papaya genetic studies and crop improvement.

Results
Identification of a candidate gene controlling YY lethality
Papaya has three sex genotypes: female (XX), hermaphrodite (XYh) and male (XY), each exhibiting distinct floral features (Fig. 1a–c). Self-pollinated hermaphrodite flowers showed a segregation ratio of 2 XYh hermaphrodites to 1 XX female, with ~25% of seeds aborted due to YhYh lethality, lacking discernible embryos (Fig. 1d, e). The aborted YhYh genotype seeds had no discernible embryos (Fig. 1f,g). In contrast, fertilizing female flowers with hermaphrodite pollen resulted in a 1 XYh hermaphrodite to 1 XX female ratio without seed abortion (Fig. 1d, e and Supplementary Table 1).

Papaya ovules exhibited retrograde development, with early and late globular embryos forming at 35 and 49 days post-pollination, respectively. Heart embryos at 65 days, followed by torpedo stage at 72 days, and cotyledon stage at 79 days (Supplementary Fig. 1). However, aborted embryos of the YhYh genotype exhibited phenotypic differences from their normal counterparts. Up to 50 days after pollination (DAP), embryos of the YhYh genotype were indistinguishable from normal XYh embryos. However, as wild-type embryos progressed to the cotyledon stage, YhYh embryos exhibited degeneration of embryo and endosperm (Fig. 1f–i).

Given the lethal phenotype observed in YY/YhYh individuals, we hypothesize that the gene responsible for YY/YhYh lethality remains functional in the X chromosome but becomes non-functional in the MSY and HSY. To investigate this, we screened 34 X-specific genes9 (Supplementary Data 1). Functional annotation identified PYhCpXYh7 encodes an ankyrin repeat domain-containing protein orthologous to Arabidopsis EMB506, which is an essential gene for normal embryo development18. Transcriptomic analysis revealed CpEMB506 is highly expressed across four embryonic stages, with its expression level gradually declining as development proceeds (Supplementary Data 1). The gene function and expression pattern made CpEMB506 as a strong candidate gene underlying YY, YYh, and YhYh lethality. The X allele features a large first intron of 8374 bp, which includes DNA transposons and LTR retrotransposons. In contrast, the HSY allele has lost the first exon (409 bp) and 7352 bp in the first intron, rendering it a pseudogene (Fig. 2a, b). A comparison of the coding DNA sequences of X, HSY, and MSY alleles revealed that the disrupted MSY and HSY alleles share conserved sequences, but differ from the X allele by SNPs and Indels (Supplementary Fig. 2). Further quantitative PCR and RNA-seq data showed that the CpEMB506-X allele is expressed in both embryos and female flowers, whereas Cpemb506-HSY pseudogene is not expressed (Supplementary Fig. 3).

Validation of CpEMB506 function and generation of viable YhYh and YYh papaya plants
To validate the function of CpEMB506, we generated transgenic plants expressing CpEMB506 under its native promoter to assess its ability to rescue the abortion phenotype in YhYh seeds. The pOX vector was transformed into papaya cultivar “Zhonghuang” (ZH) calli using an efficient genetic transformation system (Supplementary Fig. 4). Transgenic lines of ProCpEMB506::CpEMB506/ZH (CpEMB506Pro/ZH) female plants were generated and confirmed via PCR (Supplementary Fig. 5).

These transgenic females were crossed with pollen from hermaphrodite cultivars pollen, producing hermaphrodite F1 plants named transCpEMB506 XYh (ProCpEMB506::CpEMB506/F1 XYh). These F1 plants were then self-pollinated to produce a segregating F2 population, which was subsequently screened to identify YhYh genotype plants (Supplementary Fig. 6). All F2 seeds exhibited black seeds, with no presence of white aborted seeds. In contrast, the control hermaphrodite seeds showed aborted white seeds, indicating that the phenotype of aborted seeds in hermaphroditic plants was partially restored (Fig. 2c). YYh genotype was obtained by crossing CpEMB506Pro/ZH XYh with ZH XY or AU9 XY male plants (Supplementary Fig. 7).

Next, two F2 populations were developed from two test crosses for identifying YhYh and YYh genotype plants, 20–1 population derived from a cross between a “SunUp” female and a 20-1 YYh male, and 15–4 population derived from a cross between a ZH female and a 15-4 YYh male. We identified 94 YYh seedlings from 20–1 population, and 32 YYh from 15–4 population after testing 4652 and 1386 F2 individuals, respectively (Supplementary Fig. 8 and Supplementary Data 2). However, only 18 YhYh plants were identified in the 20–1 population, with no such individuals detected in the 15–4 population (Supplementary Fig. 8 and Supplementary Data 2). All YYh and YhYh were identified using the developed sex-specific CpEMB506, PYhCpXYh31 and TE markers. These markers effectively distinguish between female and hermaphrodite plants, as well as between male and hermaphrodite plants (Supplementary Tables 2 and 3). To further validate the accuracy of the YhYh genotype, we conducted next-generation resequencing on six YhYh plants and eight YYh as control. Sequencing results confirmed the absence of the X-linked sex determination region in all assayed plants (Fig. 2d). We aligned the sequencing data to the AU9 MSY genome as the reference and analyzed 15 high-confidence SNP markers located within the MSY. Genotype analysis revealed that all six YhYh samples matched the HSY genotype, verifying the accuracy of YhYh genotype identification (Supplementary Figs. 9, 10 and Supplementary Data 3). However, all 18 YhYh seedlings and 32 of the 126 YYh seedlings displayed reduced viability, with post-embryonic developmental arrest occurring in 100% of YhYh and 25.4% of YYh individuals (Supplementary Data 2). We observed that the apex of 18 YhYh and 32 YYh seedlings gradually ceased development, leaving only the stem, and eventually, the seedlings died after approximately 10 days of growth (Fig. 2e).

The YYh genotype plants exhibited a male phenotype. Comparative analysis of YYh and XY male plants showed no significant differences in plant height, flower structure, and peduncle length compared to XY males (two-tailed t-test, p=0.14–0.97) (Fig. 2f–i and Supplementary Fig. 11). Scanning electron microscopy (SEM) confirmed that pollen morphology and vitality were identical between YYh and XY male flowers (Supplementary Fig. 12).

FISH analysis revealed that the Y-chromosome-specific probe generated one strong signal in one Yh chromosome in XYh hermaphrodite “SunUp”, and two strong signals in two YYh individuals, confirming that the fertility of YYh was restored (Fig. 2j to l). These results validated that CpEMB506 is the gene controlling YY, YYh, YhYh lethality. We renamed this YY lethality gene CpEMB506 as CpYYL.

The CpYYL is expressed in embryo and encodes a chloroplast localized protein
To elucidate the physiological function of CpYYL, its expression was examined across various papaya tissues. CpYYL was expressed in leaves, roots, stems, and at two floral developmental stages of male, hermaphrodite, and female plants. Expression was markedly higher in flowers, especially in female flowers and their pistils (Supplementary Fig. 13a). The highest expression level was observed in ovules from fruit 5–15 DAP. As ovules developed, expression gradually decreased, with a sharp decline at 35 DAP (Supplementary Fig. 13b).

To further validate spatial expression patterns, we performed in situ hybridization of CpYYL in embryos. Strong hybridization signals were detected in both the early globular embryo and the endotesta at 35 DAP, whereas weaker signals were observed in globular-shaped embryo and endotesta at 50 DAP (Fig. 3a–f), indicated that CpYYL functions in early embryo development.

AtEMB506 protein is localized in the chloroplast, and has a chloroplast targeting signal peptide19. The investigation of subcellular localization of CpYYL displayed CpYYL-GFP was co-localized with the chlorophyll fluorescence signals (Fig. 3i,k), indicating a chloroplast localization. However, when we deleted the putative signal peptide of 41 amino acids directing CpYYL to plastids, CpYYL-del-GFP was irregularly distributed on the cell membrane and not co-localized with the chlorophyll fluorescence signals, confirming the existence of a signal peptide for targeting CpYYL to the chloroplasts (Fig. 3j).

The mutation of CpYYL impacts the expression of genes in sucrose metabolism
To evaluate the impact of CpYYL loss on gene expression, we conducted RNA-seq analysis on ovules at different developmental stages from crosses between “Zhongbai” XX female and XYh hermaphrodites, as well as from self-pollinated XYh hermaphrodites. Because it is not possible to distinguish aborted seeds in self-pollinated hermaphrodite fruit based on phenotype before seed maturation, we included both cross types in the analysis. A total of 50, 524, 801, and 39 DEGs detected at 5, 15, 35, and 65 DAP ovules between ovules of Zhongbai females from the cross and Zhongbai hermaphrodites from self-pollinated plants (Fig. 4a).

We identified 412 and 683 unigenes expressed at 15 and 35 DAP (Fig. 4b). Our focus was on genes specifically expressed at the 15-day stage between female and hermaphrodite ovules. Gene Ontology (GO) enrichment analysis revealed that the downregulated DEGs were mainly enriched in post-embryonic morphogenesis, succinate transport, oxidation-reduction process and hormone stimulus response. In contrast, the upregulated DEGs are primarily associated with precursor metabolites and energy, pyruvate metabolic and glycolytic processes (Fig. 4c). Further analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that the downregulated DEGs were mainly enriched in pathways related to carbon fixation via the Calvin cycle, glycolysis, and starch and sucrose metabolism (Fig. 4d).

Sucrose metabolism provide energy for embryonic development20–22. The key enzymes involved in this process, including alkaline/neutral invertases (A/N-Inv-C), which are localized in the mitochondria, and catalyzes the breakdown of sucrose into glucose and fructose, thereby fueling various cellular processes23. Additionally, a suite of other enzymes, including hexokinase 4 (HXK4), cytoplasmic phosphoglucose isomerase (CPGI), PFK3, fructose-1,6-bisphosphate aldolase (FBA6)24, fructokinase (FRK), triosephosphate isomerase (TPI), and fructose-bisphosphate aldolase 6 (FBA6)25, plays significant roles in this metabolic cascade. The genes encoding these enzymes all showed increased transcription in 15-day hermaphrodite ovules (Fig. 4e, f), which in turn accelerates the glycolytic pathway. This metabolic shift is further underscored by the observation of significantly reduced sucrose content in hermaphrodites at the 15-day stage (Fig. 4g, h, Supplementary Fig. 14, and Supplementary Data 4, 5), highlighting the dynamic changes in energy metabolism during this critical developmental phase.

CpYYL interact with CpAKRP during the papaya life cycle
To explore the molecular mechanisms underlying protein interaction involving CpYYL, we screened for potential interacting proteins. Yeast two-hybrid analysis revealed a strong interaction between CpYYL and an ankyrin repeat protein, CpAKRP (Fig. 5a, b). AKRP is crucial in the initial differentiation of the proplastid19. Domain interaction analysis using truncated domains of CpYYL and CpAKRP showed that the interaction was markedly weakened when only the N-terminal regions of both proteins were present, whereas efficient binding required the four ankyrin (ANK) domain repeats along with its upstream region (Supplementary Fig. 15).

Subsequent BiFC-assays confirmed the in vivo interaction between CpYYL and CpAKRP (Fig. 5c). Due to papaya’s two-year transformation cycle, we used transformed Arabidopsis to validate the role of CpAKRP. Overexpression of CpYYL and CpAKRP partially rescued the 25% seed abortion phenotype in the heterozygous emb506 and akrp mutants (Fig. 5d, e).

Population genomic analysis of CpYYL
To investigate the evolutionary history of YYL, we analyzed 63 sequenced plant genomes. The YYL gene predates seed plants, with orthologs identified in algae, liverworts, mosses, and ferns, the earliest evolutionary origin of YYL gene can be traced to Chlamydomonas, highlighting the early origin and evolution of YYL (Supplementary Fig. 16a). Examination of YYL across 63 gymnosperm and angiosperm genomes revealed strong conservation, particularly in the ANK repeat domain associated with protein interactions (Supplementary Data 6 and Supplementary Fig. 16b). The CpYYL-X allele demonstrated was confirmed to be functional based on its intact gene structure and complementation test. In contrast, the cpyyl-h allele is a pseudogene, lacking expression due to the absence of the first exon and part of the first intron. Nucleotide diversity (π) for CpYYL-X was 5.26e-05 in cultivated and 8.33e-05 in wild papaya, whereas for cpyyl-h, it was 2.01e-06 in cultivated and 3.48e-06 in the wild papaya (Supplementary Fig. 17a, b), indicating limited diversity.

Furthermore, the Tajima’s D value for CpYYL-X was -1.34 in cultivated and -1.15 in wild papaya, with FST scores ranging from 0 to 0.1 (Supplementary Fig. 17c–f). These values suggest a potential selection pressure acting on this allele. In contrast, cpyyl-h’s Tajima’s D value was near zero (Supplementary Fig. 17f), indicating that this pseudogene has not been subject to selection pressures due to mutations.

Recombination suppression between HSY and MSY in YYh male papaya
The creation of viable YYh male plants offered an unprecedented opportunity to identify the carpel suppressing sex determination gene in male flowers through forward genetics. “SunUp” females were crossed with 24-6 YYh males and generated a testcross population segregating males to hermaphrodites at 1:1 ratio. Male- and hermaphrodite-specific DNA markers were used to genotyping 537 testcross plants (Supplementary Fig. 18), yielding 294 male and 243 hermaphrodite genotypes, perfectly matching the sex phenotypes with zero recombinant plants (Supplementary Data 7).

We also examined the sex segregation ratio in the offspring of the two test cross 20-1 and 15-4 population (Supplementary Figs. 19 and 20). The 20-1 testcross population consisted of 194 plants, with 110 hermaphrodites and 84 males, while the 15-4 testcross population comprised 483 plants, with 236 hermaphrodites and 247 male plants. Both populations fitted to a 1:1 male to hermaphrodite segregation ratio (Chi-square test, p=0.062 for 20–1 populations and p=0.616 for 15–4 populations).

Methods
Material and conditions
The papaya seedlings, SunUp, ZH, and AU9 are preserved resources in the Center for Genomics and Biotechnology at Fujian Agriculture and Forestry University, while the Zhongbai variety was purchased from the market. The papaya seedlings were cultivated in the greenhouse at Fujian Agriculture and Forestry University, maintained at a temperature of 28°C with a 16-h light/8-hour dark photoperiod. After two months of cultivation, the seedlings were planted in the papaya field in Yongchun, Fujian, and in the experimental plots within the campus of Fujian Agriculture and Forestry University. Arabidopsis thaliana wild-type ecotype Columbia (Col), along with transgenic and mutant lines, were cultivated in MetroMix 360 soil under controlled conditions of 22°C, 55% relative humidity, and a 16-h light/8-h dark photoperiod.

Generation of transgenic plants
To explore the function of the CpYYL-X copy and to produce viable YhYh genotypes in papaya. We transformed female papaya calli with the construct ProCpYYL_X::CpYYL_X. To construct the vector ProCpYYL_X::CpYYL_X, we utilized the expression vector pOX from our laboratory. The 4.7 kb promoter region and the 990 bp CpYYL_X coding sequence (CDS) fragment were amplified by PCR and subsequently cloned into the pOX vector. The primer sequences (synthesized by Sangon Biotech, Shanghai) were as follows: Pro-CpYYLF: ACCGGAATTCATCGCGACTTGTAGGTGCCATAGT; Pro-CpYYLR: AACCCTGCAGTGATTGCCAACTTGT
TTATTCAACCCTTGTGT; CpYYLCDSF: AAAAACTGCAGATGGCCTCGTGGGCTACGACAGTT; CpYYLCDSR: CCCAACGCGTTTATGGATATTGGTCAGTTGCTATGATC. The genetic transformation procedure was primarily adapted from the protocol previously published43. Specifically, K5 medium containing 5 mg/L 2,4-D and 0.2 mg/L KT was used to induce callus formation (1/2 MS + 5 mg/L 2,4-D + 0.2 mg/L KT + 3 g/L hytagel + 30 g/L sucrose). We used the young hypocotyls of ZH tissue culture seedlings as explants. Subsequently, medium M13 (1/2 MS + 5 mg/L 2,4-D + 3 g/L phytagel + 30 g/L sucrose) was employed to promote embryogenic callus development. In the final stage, embryogenic calli were transferred to CMM (1/2 MS + 10 mg/L 2,4-D + 3 g/L phytagel + 70 g/L sucrose) medium to encourage multiplication. These multiplied embryogenic calli were then subjected to Agrobacterium-mediated transformation. Embryogenic callus induction followed a three-step method, which could significantly improve induction efficiency and shorten the induction period44,45. For the transformation process, papaya embryogenic calli were immersed in transformation buffer for 15 min, and co-cultivation on CMM medium for 24 h. Post co-cultivation, the tissues were transferred to CIM1 medium (1/2 MS + 10 mg/L 2,4-D + 3 g/L phytagel + 70 g/L sucrose) for somatic embryogenesis, with carbenicillin (200 mg/L) and cefotaxime (200 mg/L) treatments. After two weeks, the tissues were moved to selection medium MBN (1/2 MS medium with 0.2 mg/L each of benzylaminopurine (6-BA) and naphthalene acetic acid (NAA), 100 mg/L timentin, and 25 mg/L hygromycin) to select putative transgenic calli. Plantlet regeneration was achieved using MBN for shoot development and IBA rooting medium (1/2 MS with 0.2 mg/L IBA). To verify hygromycin-resistant seedlings, we utilized PCR with two pairs of primers. One pair was designed to amplify the CpYYL coding sequence. The second primer was used to amplify a segment of the CpYYL coding sequence along with the vector sequence.

Genotyping of YhYh and YYh genotype plant
The CpYYL expression vector driven by its native promoter was introduced into ZH female calli to obtain T1 generation plants. These T1 plants were then crossed with hermaphroditic plants, such as SunUp XYh, to generate the F1 population. We initially screened for CpYYL transgenic plants using two pairs of transgene verification primers previously mentioned. Next, the W11 primer was employed to identify male or hermaphroditic plants46. The screened hermaphroditic transgenic plants were subjected to self-pollination. Seeds from these self-pollinations were planted in seed trays, and once the seedlings developed three leaves, PCR identification was conducted to screen for YhYh or YYh plants. Using transgene verification primers and W11 primer, we identified transgenic plants containing Y or Yh copies. The Intron1, PYhCpXYh31-1 and PYhCpXYh31-2 primers target the X copy-specific fragment of CpYYL and PYhCpXYh31. Amplification of three fragments indicate the presence of the X chromosome, while failure to amplify suggests a YhYh or YYh genotype. The TE-1 Yh-specific primer, designed based on the TE upstream of the CpMp gene, successfully amplifies the band in Yh copy, not the Y copy. Conversely, the TE-2 Y-specific primer, positioned at the TE insertion site, amplifies a band exclusively in males, but not in hermaphrodites. These specific primers allow us to distinguish between YhYh and YYh plants.

To further verify the accuracy of YhYh plants, we conducted second-generation resequencing by illumina xplus. Duplicate and low-quality reads were filtered out using Picard tools (v.2.20.4, https://github.com/broadinstitute/picard) and Trimmomatic47 (a Java software, v0.38), respectively. Filtered reads were aligned to SunUp reference with BWA-MEM (v0.7.17-r1188)48, and the SAM output file was converted to the BAM format using the SAMtools suite49. The BAM files were visualized using IGV-GSAman (v.0.8.8) (https://gitee.com/CJchen/IGV-sRNA). By evaluating 15 high-confidence SNP markers located in the MSY and HSY region, we confirmed the YhYh genotype. Additionally, we assessed the read coverage in the X-specific region. YhYh genotype plants showed very low coverage compared to the XYh control, indicating the absence of the X copy in the verified YhYh genotype plants. Through molecular and sequencing validation, we successfully identified YhYh plants.

Design and preparation of Y-chromosome-specific oligoprobe
We used an established methodology to identify Y-chromosome-specific oligonucleotides50 within the SunUp genome2. The oligonucleotides were screened using Chorus2 software51, with the detailed protocol outlined as follows: oligonucleotides (59 nucleotides in length) unique to the SunUp genome, employing a step size of 5 nucleotides. Oligonucleotides were excluded if they mapped to multiple locations with at least 75% similarity across the genome. We chose oligonucleotides with a differential melting temperature (dTm) greater than 10°C, where dTm is defined as the melting temperature minus the hairpin melting temperature. Genomic sequenced data from SunUp was used to filter out potential repeats. Ultimately, 2499 Y-chromosome-specific oligonucleotides were selected from a 3.2 Mb region, spanning positions 0.9–4.1 Mb on the Y-chromosome, and the library was synthesized by MYcroarray (Ann Arbor, MI, USA)

The preparation of oligonucleotide probes followed the previously described protocol50. Briefly, the library underwent PCR amplification to produce a double-stranded DNA template, which was subsequently transcribed into RNA. The library was labeled through reverse transcription of the RNA using a 5′ biotin-labeled primer. Unincorporated primers and the RNA template were degraded to yield a labeled, single-stranded oligonucleotide probe.

Preparation of chromosome spreads and fluorescence in situ hybridization
Chromosome preparation followed a previously established protocol50. In brief, root tips were harvested from potted plants and pre-treated with 2 mM 8-hydroxyquinoline at 25°C for 2 h. Subsequently, the root tips were fixed overnight in a 3:1 (v/v) ethanol:glacial acetic acid solution at 25°C. After a brief rinse in distilled water, the root tips were incubated in an enzymatic solution containing 2% cellulase Onozuka-R10 (Yakult Pharmaceutical, Tokyo, Japan) and 1% pectolyase from Aspergillus niger (Sigma-Aldrich Corp., St. Louis, MO, USA). Following another rinse in distilled water, the root tips were fixed again in the ethanol:acetic acid (3:1) solution. Cells were dissociated by gently breaking the root tips with a pipette tip, and the resulting cell suspension was applied to glass slides. The slides were air-dried and stored at −20°C until further use.

FISH was performed as previously described in “Method”52. The procedure is that hybridization signals were visualized using Alexa Fluor 488 streptavidin at a dilution of 1:100 (Thermo Fisher Scientific, Waltham, MA, USA) for biotin-labeled probes and rhodamine-conjugated anti-digoxigenin at a dilution of 1:200 (Roche Diagnostics, Basel, Switzerland) for digoxigenin-labeled probes. Chromosomes were counterstained with DAPI (2 mg/mL) and mounted in Vectashield (Vector). Metaphase plates with fluorescent signals were observed using an Olympus BX63 fluorescent microscope, photographed with an Olympus DP80 CCD camera, and analyzed with cellSens Dimension 1.9 software (Olympus Corporation, Tokyo, Japan).

Phenotypic observation of the pollen from YYh, ZH XY, and AU9 XY flowers
We collected fresh pollen from ZH YYh, AU9 YYh, and AU9 XY flowers, selecting those that had just bloomed for their active and easily collectible pollen. The pollen was carefully extracted and evenly distributed onto a microscope slide to ensure uniform observation. The pollen was added 2–3 drops of Alexander stain, and then covered with a coverslip to prevent evaporation and ensure even staining. After 5 h of staining, the slide was photographed with a microscope Axioimage A2 1701943S (Carl Zeiss, Oberkochen, Germany) to observe the stained pollen’s shape, size, and surface features. For a detailed structural and morphological analysis of pollen, we securely placed fresh pollen onto a slide with double-sided tape and observed it using a Hitachi TM3030 PLUS scanning electron microscope (SEM) (Hitachi, Tokyo, Japan).

Paraffin section and hybridization in situ
For paraffin section analysis, seeds of Zhongbai papaya variety were collected at 35, 42, 49, 65, 72, and 79 DAP and fixed in FAA (formaldehyde–acetic acid–ethanol) solution. The samples underwent dehydration through a graded ethanol series, followed by treatment with ethanol and xylene solutions for clearing. Subsequently, the samples were infiltrated with paraffin wax, embedded, and sectioned at a thickness of 8 μm using a Leica RM2235 microtome. Sections were stained with a 1% hematoxylin-eosin solution (dissolved in 95% ethanol) for 30 min. After staining, 1–2 drops of mounting medium were applied to the glass slide. Observations were made under microscope Axioimage A2 1701943S (Carl Zeiss, Oberkochen, Germany). The above methods were referenced from literature and modified accordingly53.

For hybridization in situ, hermaphrodite ovules of the Zhongbai cultivar were harvested at 35, 50, 65, 75, 85, and 115 DAP and prepared for paraffin sectioning as previously described54. The dehydrated samples were embedded in paraffin and sectioned at 8 μm.

Probe preparation followed kit instructions (Roche DIG RNA labeling kit SP6/T7), with T7 promoter sequences added as a 5’-tail on the reverse primer for probe region amplification (pCRII-TOPO, Invitrogen). Target DNA was reverse-transcribed to mRNA using a DIG-UTP labeling kit (Roche DIG RNA labeling kit SP6/T7), and probe length was reduced to 150 bp by fragmentation.

Tissue sections were deparaffinized and rehydrated through a graded ethanol series. Acetic anhydride solution was prepared immediately before use, and protease was added at the time of use. Hybridization probes were used at a final concentration of 0.5 ng/μl probe complexity. For hybridizations performed with 100 μl per slide, approximately 25 ng of probe was used per slide for 0.5-kb probes and 50 ng per slide for 1-kb probes. Slides were incubated at 55°C for 16–20 h. Following hybridization, slides were incubated with anti-digoxigenin antibody diluted in washing buffer (1:5 v/v) in the dark for 2–3 h at room temperature or overnight at 4°C. Sections were photographed by using NI-U research grade upright microscope (Nikon Corporation, Tokyo, Japan).

RNA-seq analysis
The paired-end RNA-seq sequences were generated using the Illumina xplus sequencing platform. Initially, quality control was performed using Fastp55 to ensure high-quality sequences. The clean reads were then aligned to the papaya reference genome2 using HISAT2 (v.2.2.1)56, a fast and sensitive alignment tool. The alignment results, initially in SAM format, were sorted and converted into BAM format using Samtools (v.1.19)57 for efficient storage and processing.

Subsequently, FeatureCounts58 was employed to quantify gene expression by counting the reads mapped to genomic features. Differential expression analysis was conducted using DESeq2 (v.1.42.0)59, with criteria set to LogFC>1 and Padj<0.05, to identify DEGs. To explore the functional implications of the DEGs, NR annotations were utilized, and GO enrichment and KEGG enrichment were conducted using the OmicShare online platform. These analyses provided insights into the biological processes and functional capabilities of the genes across different conditions.

Subcellular localization assays
The open reading frames (ORFs) of full-length CpYYL-X, and CpYYL-X-2F (remove signal peptide of CpYYL-X, residues were from 111 to 329) were inserted into the pMDC202-GFP vector to produce the pMDC202-CpYYL-X-GFP and pMDC202-CpYYL-X-2F-GFP recombinant expression vectors, then they were separately introduced into Agrobacterium tumefaciens strain GV3101 using the freeze-thaw method60. Nicotiana benthamiana leaves were prepared for transient transformation via leaf infiltration with a solution of Agrobacterium cells. After 48 h, GFP fluorescence signals were observed under a confocal laser-scanning microscope (SP8X, Leica, Germany).

Bimolecular fluorescence complementation assay
The full-length coding sequence of CpYYL or CpAKRP without the stop codon was cloned into the vector SPYNE to generate p35S CpYYL/CpAKRP-YFPN vector. The full-length coding sequences of CpYYL and CpAKRP were cloned into the vector SPYCE by In-Fusion cloning to generate p35S CpYYL/CpAKRP-YFPC vector, respectively, by using a ClonExpress Ultra One Step Cloning Kit (Vazyme Biotech Co., Ltd). The different combinations of YFPN and YFPC fusion vectors were coinfiltrated into tobacco leaf epidermal cells. Agrobacterium tumefaciens strain GV3101 cells harboring the above different constructs were cultured to OD600=0.8, then centrifuged, and the pellet was resuspended in infiltration buffer (1 mM methyl ester sulfonate, 10 mM MgCl2, and 150 mM acetosyringone, pH 5.7). Different combinations of vectors were mixed equally and coinfiltrated into leaves from 3-week-old Nicotiana benthamiana. Then they were used to detect the signal of reconstituted YFP fluorescence by a confocal laser-scanning microscope (SP8X, Leica, Germany) in the dark for 48 h after infiltration61.

RNA extraction and quantitative real-time PCR (qPCR)
In order to analyze the expression of genes, total RNA was extracted using the RNA Isolation Kit (TAKARA). The reverse transcription of total RNA was performed with PrimeScript TM RT reagent Kit, which includes gDNA Eraser (TAKARA). qPCR was conducted using TB Green SYBR Premix Ex Taq TM II (Tli RNaseH Plus) (TAKARA). qPCR was carried out using a CFX real-time PCR system (BioRad). Data from three biological replicates were used to calculate expression levels using the 2−△△CT method62. CpActin was used to normalize the RNA levels between different samples. Analysis of variance (ANOVA) was used to test the significance of expression level. Three technical replicates for each sample were performed for qPCR analysis.

Yeast screening assay
For pGBKT7 (Clontech) bait vectors, the truncated coding sequences of CpYYL were fused down-stream of the GAL4 DNA binding domain (BD). The cDNA library of the papaya samples from flower, leaf and immature fruit was constructed using a Gateway approach by Ouyi Company. Two-Hybrid Library Screening is using Yeast Mating utilizing the natural ability of haploid yeast strains as Y187 and Y2H Gold to mate with each other to form a diploid cell. Spread the mated culture on the triple dropout supplements (TDO) plates. High stringency patching to identify potential binding partners on the quadruple dropout supplements (QDO) plates (Clontech). Positive cloned yeast plasmids were extracted and sequenced. After sequencing the positive clones, the sequence results were blasted by NCBI and got to the target sequence. Then, Yeast Two-Hybrid was chosen to further confirm each screening protein–protein interaction.

Yeast two-hybrid assay
For pGBKT7 (Clontech) bait vectors, the truncated coding sequences of CpYYL were fused downstream of the GAL4 DNA-binding domain to generate the BD recombinant plasmid. For pGADT7 (Clontech) prey vectors, the full-length coding sequences of CpAKRP were fused to downstream of the GAL4 activation domain to generate AD recombinant plasmid. Different combinations of BD and AD constructs were co-transformed into the yeast strain Y2HGOLD using a Frozen-EZ Yeast Transformation II Kit (MKbio). The manipulations were performed according to the manufacturer’s instructions (Clontech) and previous studies63. The interaction between pGBKT7-53 (Human P53) and pGADT7-T (T-antigen) was used as a positive control. The interactions between pGADT7-empty and pGBKT7-empty were used as a negative control. Co-transformants were subsequently spotted on SD/-Leu/-Trp medium (DDO) and to assess protein interactions, the positive yeast clones were spotted onto SD/-Ade/-His/-Leu/-Trp medium (QDO) with a series of dilution concentrations. The Interactions results were observed after incubation at 30°C after 3 days.

Arabidopsis genetic transformation analysis
The emb506 (SALK_054289C) T-DNA insertion mutant was sourced from Arashare, while the akrp (CS16042, CS26041) T-DNA insertion mutant was generously provided by Lixin Zhang’s lab at Henan University. Plasmid constructs were generated using the ClonExpress II One Step Cloning Kit (Vazyme). For transgenic plant generation, the cDNA of CpYYL was inserted into the XbaI and SalI sites of the pMDC202 vector, and the cDNA of CpAKRP was inserted into the XhoI and SalI sites of the pGWB505 vector. These constructs were subsequently introduced into Agrobacterium tumefaciens strain GV3101.

Heterozygous seeds of emb506 and akrp mutants were cultivated to flowering, after which the overexpression constructs were introduced via the floral dip method64. Transgenic plants were selected on 1/2 MS medium supplemented with 30 µg/mL hygromycin and 50 µg/mL kanamycin. Successful integration of the transgenes in the T1 generation was confirmed by PCR. Homozygous transgenic lines were identified in the T3 generation, and two lines were selected for further analysis. For each line, at least 30 siliques were harvested to assess sterility rates.

GC-MS analysis of sugars
Freeze-dried papaya ovules from both female and hermaphroditic plants at 15, 35, and 65 days DAP (total 16 samples) were pulverized using a mixer mill (MM400, Retsch) equipped with a zirconia bead, operating at 30 Hz for 1.5 min, three biological replicates were performed. A 20 mg portion of the resulting powder was diluted to 500 μL with a methanol:isopropanol:water mixture (3:3:2, v/v/v), followed by vortexing for 3 min and sonication for 30 min. A 50 μL aliquot of the supernatant was combined with 20 μL of an internal standard solution (ribitol, 1000 μg/mL) and evaporated under a nitrogen gas stream. The dried sample was then subjected to freeze-drying using a lyophilizer, and the residue was prepared for derivatization.

For derivatization, the sample was treated with 100 μL of methoxyamine hydrochloride in pyridine (15 mg/mL) and incubated at 37°C for 2 h. Subsequently, 100 μL of BSTFA was added, and the mixture was vortexed and maintained at 37°C for an additional 30 min. The derivatized sample was then diluted to an appropriate concentration for GC-MS analysis.

GC-MS analysis was performed using an Agilent 8890 gas chromatograph coupled to a 5977B mass spectrometer, equipped with a DB-5MS column (30 m length × 0.25 mm i.d. × 0.25 μm film thickness, J&W Scientific, USA). Helium was used as the carrier gas at a flow rate of 1 mL/min. Samples were injected in split mode with a split ratio of 5:1, and an injection volume of 1 μL. The oven temperature program was as follows initial hold at 170°C for 2 minutes, ramp to 240°C at 10°C/min, further increase to 280°C at 5°C/min, and a final increase to 310°C at 25°C/min, with a hold for 4 min. All analyses were conducted in selective ion monitoring mode, with the ion source and transfer line temperatures set at 230°C and 240°C, respectively65,66. Sugar contents were quantified by MetWare (http://www.metware.cn/) using the Agilent 8890-5977B platform.

Statistical analysis was conducted in the R environment, with differentially accumulated metabolites screened using a Fold Change|≥2 threshold, and pathway enrichment analysis performed using a one-tailed hypergeometric test.

Multiple sequence alignment and tree construction
Sequences of EMB506 proteins, annotated from transcripts and genomes, were aligned using MUSCLE (v.5.1)67 with default parameters. The regions poorly aligned were trimmed using trimAl (v.1.4)68 and the trimmed alignments were used for the construction of a maximum likelihood tree using IQ-TREE (v.2.1.4)69 under the optimal substitution model. For each tree, 1000 replicates were conducted to generate bootstrap values. The trees were viewed using iTOL (v6)70, while Jalview was utilized for viewing the alignment results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the present manuscript, the genes and genomic architecture in the sex-determining regions (SDRs) that control sex segregation and characteristics of each sex were identified in papaya. Particularly, it is noteworthy that the authors succeeded in identifying the gene responsible for lethality in Y or Yh homozygotes and the successful production of Y (Yh) homozygous individuals by complementation with overexpression of the causal gene. Also, it was novel and unique works that mutants showing the sex reversal phenotype were screened and identified. Each study was excellent and quite informative for elucidating structure and functions of papaya sex chromosomal regions.\nAlthough there are multiple unique and novel discoveries and efforts made in this study, first, the objective of the study in this paper is not clearly stated. According to the results in this paper, multiple targets for the studies seem to be present in this study. Of course, they are related to sex chromosome (Y, Yh) in papaya, but I could not understand the identity of the study in this paper. Owing to this, it was impressed that identified findings shown in the result section were quite dispersed.\nRegarding to each specific result,\nFirstly, I could not find how the CpEMB506 gene was identified or regarded is as the candidate for YY lethality gene in papaya? Furthermore, the X allele of CpEMB506 is described as functional allele, but its evidences were not shown in this study. The result that YhYh individuals were successfully developed in the CpEMB506-OX progeny is the quite impressive and novel result, but there seems to be no detail of their phenotypes.\nOn the other hand, whole genome sequencing of transgenic individuals of CpEMB506 (CpYYL)-OX plants was performed to determine the position of transgene insertions, but I am not sure how these results contribute to the main story or objectives in this paper.\nYhYh and YYh plants derived from CpEMB506-OX individuals are certainly helpful for determining the HSY and MSY sequences and finding their structural differences systematically, but it is not always worth enumerating each structural variations in p7, because further analyses of these variations were not carried out in this paper.\nNext, it is also unclear why the authors focused on CpMp gene in this study. It seems that the functional elucidation of CpMp will be shown in another publication, but its citation seems to be shown, but it was insufficient (no statement on publication status). Since the details of the revised article for CpMp gene study were unknown, I am not sure if there is any overlap with the data and description presented in this paper. It was essential for authors to discriminate the results and experimental data between present paper and another manuscript under review for avoiding replicate or duplicate publications.\nObtained sex reversal mutants must be quite useful materials for identifying genes and elucidating the mechanism of papaya sex determination. But, it seemed to be waste of these sex reversal mutants to demonstrate that CpMp gene was not likely to be the candidate genes for sex determination. Is it necessary for this study??\nAgain, topic of CpYYL appeared in the latter part of results section without any continuity to previous descriptions, so it should be described in an organized manner.\nIn the discussion, related to the title of this paper, definition or meaning of “domestication” in papaya should be well considered.\nI suppose that the domestication of papaya (Carica papaya) is the emergence of trioecy (hermaphrodite), and/or increased fruit size in hermaphrodite in comparison with female fruit. The YY lethality by CPYYL gene as described in this paper was suggested to contribute to the establishment of dioecy in the papaya (or its ancestors) before domestication. CpMp is suggested to be the gene for peduncles length. Though there are differences in peduncle length between male and hermaphrodite plants, I am not sure how change of peduncle length affects the domestication of papaya.\nAs the additional issues,\nYY, YhYh, and YYh lethality should be considered as the same phenomena? So far as present manuscript, these words seemed to be co-existed in the text.\nIn discussion, what does \"super hermaphrodite YhYh plant\" mean in its phenotype?\nIn Fig4k, show pictures of female flowers rather than female fruits.", "reviewer_2": "This manuscript by Yue et al. addresses an important question on sex determination in the trioecious papaya species. The paper deciphers the mechanism behind the lethality of the fourth YY sex combination, identifying the YY lethality gene CpYYL (CpEMB506). The work demonstrates that the absence of the CpYYL gene in the Y chromosome is the causal mechanism for the observed lethality of the YY genotype, notably by obtaining viable YY individuals via expression of the CpYYL gene. The authors provid a wealth of experiments and evidences to support their conclusions. This is a comprehensive and very interesting work combining a multitude of approaches (molecular genetics, functional genomics, functional validation, ...) that will be of interest to a wide scientific community, beyond those working on papaya. The work is convincing and opens up new prospects for papaya breeding.\nI have a few minor comments and few suggestions to make to the authors. The manuscript also contains typos and grammatical errors, some of which are listed below:\n- 101: The successful assemble of the asparagus YY genome …”\nshould read the “The successful assembly of the asparagus YY genome…”\n- 143-149 and Mat & Methods: Somatic embryogenesis. There are few published papers that describes somatic embryogenesis using papaya hypocotyl, and genetic transformation and regeneration. I suggest that the authors describe whether their protocol is derived from one of these articles and what improvements they have made to the present\n- 152: “to producing” should read “to produce”\nLine 215: Which population does YYh comes from? Looking at the Hi-C heatmap data (Supplementary Fig. 21), it appears to come from genotype 15-4YYh. It would be helpful to indicate this information in the manuscript.\n- 364-365: The sentence is unclear. Suggestion: “We focused on genes specifically expressed at the 15-day stage between two samples.”\n- 370-375: check the grammar\n- 470: CpYYL was .... should Read CpYYL is ….\n- Line 477-481: Something is missing in the sentence “Utilizing a complementation strategy where the emb506 and akrp mutants in Arabidopsis were partially complemented by overexpressing CpYYLand CpAKRP gene, respectively”. Please rewrite\n- Line 1108: The NCBI accessions are missing and are marked as (XX)\n- Materiel & Method: The papaya cultivar used in paraffin sections (Figure1h,i ; Supplementary Figure2) is not described.\n- Figure1a,c: The photos show that the male flower has 5 stamens and the hermaphrodite has 10 stamens, but the inverse is written in the figure legend?\n- Figure3a: The color code is not described. Is it the same as in “f”. Please precise in the. Figure and/or I the legend.\n- Supplementary Figure19c: in the legend, I believe that “SunUp female and YYh20-1, 20-1XYh (15-4H) and 20-1XY (15-4M) were selected for further experiments” should read :\nSunUp female and YYh20-1, 20-1XYh (20-1H) and 20-1XY (20-1M) were selected for further experiments”.\n- It will be helpful to people not in the papaya community, to mention in the beginning of the main text, the ploidy level and chromosome number of papaya.\n- Discussion section: I suggest that the authors discuss the link between CpYYL and sugar content and how CpYYL impacts sucrose metabolism?", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The manuscript \"Recreating viable YhYh genotype uncovers a domestication event of the Yh chromosome in papaya\" presents a groundbreaking study on the domestication and genetic basis of sex determination in papaya with a focus on the Y chromosome. Key findings include the identification of the YY lethality gene (CpYYL) and its role in embryo viablity as well as the discovery of a retrotransposon insertion in the CpMp-h promoter as a crucial domestication event. Moreover, the authors successfully generated viable YhYh and YYh genotypes, addressing a long-standing challenge in papaya breeding. Although papaya is not a classical easily manipulated lab model, the authors provide strong functional validation of the genes studied. The figures effectively illustrate key results, such as FISH validation, structural variants, and methylation patterns.\nHowever, the manuscript is dense particularly in the Results section. Reorganizing for better readability — such as adding subheadings or including a summary figure of the experimental workflow — would be helpful.\nSome terms (e.g., \"stage 3 sex chromosomes\") are introduced without sufficient context for non-specialists.\nThe partial lethality of YhYh/YYh genotypes (59.6% and 26.9% mortality) is attributed to missing X-specific genes but lacks mechanistic detail. It would be beneficial to discuss whether this lethality results from haploinsufficiency or specific gene interactions.\nFor improved clarity, adding a schematic model summarizing the molecular mechanism of YY lethality, CpYYL function, and the domestication of Yh is recommended.\nDespite these comments, the manuscript represents a significant contribution to understanding the evolution and domestication of papaya, and I recommend it for acceptance after revisions."}, "author_response": "Point by point response to reviewers’ comments\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn the present manuscript, the genes and genomic architecture in the sex-determining regions\n(SDRs) that control sex segregation and characteristics of each sex were identified in papaya.\nParticularly, it is noteworthy that the authors succeeded in identifying the gene responsible for\nlethality in Y or Yh homozygotes and the successful production of Y (Yh) homozygous\nindividuals by complementation with overexpression of the causal gene. Also, it was novel and\nunique works that mutants showing the sex reversal phenotype were screened and identified. Each\nstudy was excellent and quite informative for elucidating structure and functions of papaya sex\nchromosomal regions.\nAlthough there are multiple unique and novel discoveries and efforts made in this study, first,\nthe objective of the study in this paper is not clearly stated. According to the results in this paper,\nmultiple targets for the studies seem to be present in this study. Of course, they are related to sex\nchromosome (Y, Yh) in papaya, but I could not understand the identity of the study in this paper.\nOwing to this, it was impressed that identified findings shown in the result section were quite\ndispersed.\nResponse: Thank you very much for your assessment and suggestions. The objectives of this\nstudy are to identify the gene controlling YY lethality and to restore viable YhYh hermaphrodite\nfor breeding true breeding hermaphrodite cultivars to solve the long-standing problem of sex\nsegregating seedlings. We have identified and validated the X-specific gene CpYYL controlling\nYY lethality, and created viable YhYh hermaphrodites, which are being used in our papaya\nbreeding program. We have added these two objectives at the end of the introduction section.\nIn the process of assembling and analyzing the haplotype resolved YYh genome, we\nidentified a retrotransposon insertion in the promoter region of the long male peduncle gene,\nCpMp, which led to the reversion of the long male peduncles into short hermaphrodite peduncles\nin papaya. This event plays a crucial role in the domestication of hermaphrodite papaya from a\ndioecious population in Central America about 4,000 years ago.\nHowever, this finding is not relevant to our objectives in this study. As per your suggestion,\nalthough each finding is significant, the presence of multiple research outcomes made the paper\nappear less focused. Therefore, we have restructured the manuscript to focus primarily on the in-\ndepth analysis of the CpYYL gene function. We have removed the sections on the CpMp gene and\nthe sex-reversal mutants to focus on the study's main objectives.\nRegarding to each specific result, firstly, I could not find how the CpEMB506 gene was identified\nor regarded is as the candidate for YY lethality gene in papaya?\nResponse: We have added how we identified the candidate gene and revised relevant text as\nbelow:\n1\n“Given the lethal phenotype observed in YY/YhYh individuals, we hypothesize that the gene\nresponsible for YhYh lethality remains functional in the X chromosome but becomes non-\nfunctional in the MSY. To investigate this, we screened for X/Y gene/pseudogene pairs located in\nthe MSY and its X counterpart, focusing on those implicated in embryonic development. This\nanalysis identified CpEMB506, an X- specific gene with a Y-pseudogene allele. Its ortholog in\nArabidopsis is associated with embryo abortion16, making CpEMB506 as a strong candidate gene\nunderlying YhYh lethality. The X allele features a large first intron of 8374 bp, which includes\nDNA transposons and LTR retrotransposons. In contrast, the HSY allele has lost the first exon\n(409 bp) and 7352 bp in the first intron, rendering it a pseudogene (Fig. 2a and b). A comparison\nof the coding DNA sequences of X, HSY, and MSY alleles revealed that the disrupted MSY and\nHSY alleles share conserved sequences, but differ from the X allele by SNPs and Indels\n(Supplementary Fig. 3).”\nReferences\nAlbert, S., Després, B., Guilleminot, J., Bechtold, N., Pelletier, G., Delseny, M., and Devic, M.\n(1999). The EMB 506 gene encodes a novel ankyrin repeat containing protein that is\nessential for the normal development of Arabidopsis embryos. Plant J 17, 169-179.\nWang, J., Na, J.K., Yu, Q., Gschwend, A.R., Han, J., Zeng, F., Aryal, R., Vanburen, R.,\nMurray, J.E., and Zhang, W. (2012). Sequencing papaya X and Yh chromosomes\nreveals molecular basis of incipient sex chromosome evolution. Proc Natl Acad Sci U S A\n109, 13710-13715.\nFurthermore, the X allele of CpEMB506 is described as functional allele, but its evidences were\nnot shown in this study.\nResponse: Thank you very much for pointing that out. In the transgenic experiment, we expressed\nthe CDS sequence of the X allele of CpEMB506 using its native promoter. This successfully\nrescued the embryonic lethal phenotype of the YhYh genotype, enabling YhYh individuals to\nsurvive (Fig.2c-h). This complementation provides the most direct and compelling evidence that\nthe X allele is functional, as the CpEMB506_X protein can support normal embryonic\ndevelopment. Consistent with this, the X allele encodes a complete protein with intact functional\ndomains , whereas the Yh allele is predicted to produce a truncated protein (Fig.2a-b). Moreover,\nthe normal presence of the X allele in female XX plants and their fertility further corroborate its\nfunctional role.\nThe result that YhYh individuals were successfully developed in the CpEMB506-OX progeny is\nthe quite impressive and novel result, but there seems to be no detail of their phenotypes.\nResponse: At the time of manuscript submission, the screened YhYh plants had been growing for\nthree months and had not yet flowered. Their seedlings showed no visible differences from XYh\nplants. Now that the YhYh plants have flowered, we confirmed that they produce normal\nhermaphroditic flowers (Fig.2d-h). Pollen viability assays showed that YhYh plants have\ncomparable pollen viability to XYh plants (Supplementary Fig.12b-c). Plant height and flower\n2\nlength also did not differ significantly between the two genotypes (Fig. 2i and Supplementary\nFig.12a). These results indicate that the surviving YhYh plants exhibit normal growth,\ndevelopment, and fertility to XYh plants.\nOn the other hand, whole genome sequencing of transgenic individuals of CpEMB506 (CpYYL)-\nOX plants was performed to determine the position of transgene insertions, but I am not sure how\nthese results contribute to the main story or objectives in this paper.\nResponse: Thank you very much for your thoughtful question. Initially, we performed whole\ngenome sequencing on transgenic CpYYL plants to investigate whether of transgene insertion sites\ninfluenced the ability to obtain viable YhYh progeny. While some transgenic lines successfully\nproduced viable YhYh individuals, others did not. Our analysis showed that the four insertion sites\nwere located in intergenic regions, with only one event within a gene, which did not affect its\nexpression. We found no evidence that the viability of YhYh is directly linked to the transgene\ninsertion sitesBecause these findings do not contribute to the primary focus of the CpEMB506\nfunctional study, we decided to remove this section to maintain a clear and focused narrative.\nYhYh and YYh plants derived from CpEMB506-OX individuals are certainly helpful for\ndetermining the HSY and MSY sequences and finding their structural differences systematically,\nbut it is not always worth enumerating each structural variations in p7, because further analyses of\nthese variations were not carried out in this paper.\nResponse：Thank you very much for your suggestions. Following your expert advice, we have\nrevised the manuscript by removing the sections on YYh genome assembly and structural\nvariations between the Y and Yh chromosomes. We plan to address these topics in greater depth in\nfuture studies.\nNext, it is also unclear why the authors focused on CpMp gene in this study. It seems that the\nfunctional elucidation of CpMp will be shown in another publication, but its citation seems to be\nshown, but it was insufficient (no statement on publication status). Since the details of the revised\narticle for CpMp gene study were unknown, I am not sure if there is any overlap with the data and\ndescription presented in this paper. It was essential for authors to discriminate the results and\nexperimental data between present paper and another manuscript under review for avoiding\nreplicate or duplicate publications.\nResponse: A Ph.D. student in our lab conducted an in-depth functional study of the CpMp gene,\nrevealing that while CpMp regulates peduncle length, it does not influence sex determination.\nFurther investigation showed that CpMp promotes peduncle elongation by directly activating\nCpYUC6, an auxin biosynthesis gene, thereby increasing IAA levels. Elevated IAA enhances GA,\ntriggering the expression of the GA-stimulated gene CpGASA6, which drives cell division and\nelongation. Overexpression of CpGASA6 confirmed its role in peduncle growth but revealed a\nfeedback inhibition on IAA biosynthesis (Zhou et al., 2025). This research is detailed in a\nmanuscript titled \"Identification and Molecular Analysis of the Y-specific CpMp Gene Controlling\nCommunications.\nWe ensure that there is no data overlap between these two studies. During haplotype\nassembly of YYh and structure variation analysis of MSY and HSY, we discovered a 4691 bp\ntransposon insertion associated with the promoter of CpMp gene, a key domestication event\nleading to the transformation of the long peduncles of male flowers into the short peduncles of\nhermaphroditic flowers. In contrast, Zhou et al.'s article primarily elucidates the biological\nfunction and regulatory network of the CpMp gene.\nBased on your suggestions, we have reorganized the entire manuscript to improve its clarity\nand focus. We removed the CpMp gene section to better highlight the main objectives of the study.\nBelow is the abstract of the article by Zhou et al.\nPapaya is a fruit crop possessing XY sex chromosomes. The development of long male peduncles\n(Mp) is pivotal for the evolutionary transition from gynodioecy to dioecy. The gene, CpMp, which\ncontrols peduncle length, is one of the four genes contributing to the evolution of stage 3 sex\nchromosomes in papaya. We identified a Y-specific SVP paralog, CpSVP-Yp, as a candidate gene\nfor CpMp through comparative genomic analyses and functional validation by complementation\ntests. CpMp promotes peduncle elongation by directly activating CpYUC6, an auxin biosynthesis\ngene, thereby increasing IAA levels. Elevated IAA in turn enhance GA, triggering expression of\nthe GA stimulated gene CpGASA6, which drives cell division and elongation. Overexpression\nCpGASA6 confirmed its role in peduncle growth but revealed a feedback inhibition on IAA\nbiosynthesis. Two upstream regulators of CpMp were identified, CpTRAB1, a positive regulator\nrepressed by GA, and CpGATA8, a negative regulator upregulated by CpMp, forming two\nfeedback loops. Notably, male trees produce 400 times more pollen than hermaphrodites, offering\na clear fitness advantage. In papaya, three genes - two sex determination genes and this long male\npeduncle gene, are essential for the establishment of dioecy and the initiation of sex chromosome\nevolution. These findings fill a major gap in understanding papaya sex chromosome evolution and\noffer valuable genetic resources for papaya improvement.\nReference\nZhou, Y., et al. (2025). Identification and molecular analysis of the Y-specific CpMp gene\ncontrolling long male peduncles in papaya.\nObtained sex reversal mutants must be quite useful materials for identifying genes and elucidating\nthe mechanism of papaya sex determination. But, it seemed to be waste of these sex reversal\nmutants to demonstrate that CpMp gene was not likely to be the candidate genes for sex\ndetermination. Is it necessary for this study??\nResponse: Agree. We have removed this section.\nAgain, topic of CpYYL appeared in the latter part of results section without any continuity to\nprevious descriptions, so it should be described in an organized manner.\n“The CpYYL is expressed in embryo and encodes a chloroplast localized protein”, “The mutation\nof CpYYL impacts the expression of genes in sucrose metabolism”, “CpYYL interact with\nCpAKRP during the papaya life cycle” and “Population genomic analysis of CpYYL” after the\nsection on “Validation of CpEMB506 function and generation of viable YhYh and YYh papaya\nplants.” This arrangement groups all the CpYYL-related content together, making it more logical.\n1. In the discussion, related to the title of this paper, definition or meaning of “domestication” in\npapaya should be well considered.\nI suppose that the domestication of papaya (Carica papaya) is the emergence of trioecy\n(hermaphrodite), and/or increased fruit size in hermaphrodite in comparison with female fruit. The\nYY lethality by CpYYL gene as described in this paper was suggested to contribute to the\nestablishment of dioecy in the papaya (or its ancestors) before domestication. CpMp is suggested\nto be the gene for peduncles length. Though there are differences in peduncle length between male\nand hermaphrodite plants, I am not sure how change of peduncle length affects the domestication\nof papaya.\nResponse: The CpMp gene is a critical sex-determining factor, known as the male peduncle,\nresponsible for the long, ramified, many-flowered cymose inflorescence of male trees (Storey,\n1953). The emergence of hermaphroditic flowers is a pivotal event in papaya domestication\n(Vanburen et al., 2015), yet alterations in peduncle length may represent an adaptive trait that\nevolved concurrently with domestication. The retention of long peduncles in male flowers is\nprimarily advantageous for increasing pollen production and enhancing pollen dispersal.\nConversely, when male flowers are domesticated into hermaphroditic flowers, long peduncles can\npose a challenge by increasing the distance required for nutrient transport. Previous studies have\nshown that the structural properties significantly influence fruit pedicel (FP) traits such as length,\nweight, and diameter, directly contributing to the development of high-quality fruits in cucumber\n(Che and Zhang, 2019). Fruit deformation can occur when the pedicel is excessively long or too\nshort (Yane et al., 2024). Additionally, shorter, sturdier peduncles may offer better support for\ndeveloping hermaphroditic fruits, mitigating the risk of fruit drop due to gravity or adverse\nweather conditions like wind and rain. Consequently, under the continuous selective pressure\nexerted by humans for high-yield, large-fruited hermaphroditic plants, individuals with relatively\nshorter peduncles may gain an advantage in the efficiency of assimilate transport to the fruit,\nleading to their indirect selection and eventual fixation. The variations in peduncle length\nregulated by the CpMp gene may illustrate how papaya has optimized resource allocation and\nreproductive strategies for different sexes during domestication, with male flowers focusing on\nefficient pollination and hermaphroditic flowers on efficient fruiting.\nReferences\nChe, G., and Zhang, X. (2019). Molecular basis of cucumber fruit domestication. Curr Opin\nPlant Biol 47, 38-46.\nStorey, W. (1953). Genetics of papaya. The Journal of Heredity 44, 70-78.\nVanburen, R., Zeng, F., Chen, C., Zhang, J., Wai, C.M., Han, J., Aryal, R., Gschwend, A.R.,\nWang, J., and Na, J.K. (2015). Origin and domestication of papaya Yh chromosome.\nYane, S., Zhuo, D., Chengzhen, S., Dun, B., and Haonan, C. (2024). Cytokinin-related genes\nregulate cucumber fruit pedicel length. Sci Rep 14, 024-75186.\nAs the additional issues,\nYY, YhYh, and YYh lethality should be considered as the same phenomena? So far as present\nmanuscript, these words seemed to be co-existed in the text.\nResponse: Yes. Yh was selected from the Y chromosome about 4,000 years ago, and they carry the\nsame CpYYL truncated allele, encoding a non-functional truncated protein.\nIn discussion, what does \"super hermaphrodite YhYh plant\" mean in its phenotype?\nResponse: The new term “super hermaphrodite YhYh plants” is following the term “YY\nsupermale” used in the sex chromosome research community, resulting 100% of their offsprings\nbeing hermaphrodite or male. We revised the sentence in Discussion as blow to clarify this term:\n“Crossing female parents with super hermaphrodite YhYh plants yield 100% XYh\nhermaphrodite seeds, effectively eliminating sex segregation, whereas cross female parents with\nregular XYh hermaphrodite plants yielded 50% females and 50% XYh hermaphrodites.”\nIn Fig4k, show pictures of female flowers rather than female fruits.\nResponse: Thank you very much for pointing out this issue. We have reorganized the manuscript\nto enhance its clarity and focus, and we have removed the section on sex-reversal mutants to better\nemphasize the main objectives of the study.\nReviewer #2 (Remarks to the Author):\nThis manuscript by Yue et al. addresses an important question on sex determination in the\ntrioecious papaya species. The paper deciphers the mechanism behind the lethality of the fourth\nYY sex combination, identifying the YY lethality gene CpYYL (CpEMB506). The work\ndemonstrates that the absence of the CpYYL gene in the Y chromosome is the causal mechanism\nfor the observed lethality of the YY genotype, notably by obtaining viable YY individuals via\nexpression of the CpYYL gene. The authors provid a wealth of experiments and evidences to\nsupport their conclusions. This is a comprehensive and very interesting work combining a\nmultitude of approaches (molecular genetics, functional genomics, functional validation, ...) that\nwill be of interest to a wide scientific community, beyond those working on papaya. The work is\nconvincing and opens up new prospects for papaya breeding.\nI have a few minor comments and few suggestions to make to the authors. The manuscript also\ncontains typos and grammatical errors, some of which are listed below:\n- 101: The successful assemble of the asparagus YY genome …”\nshould read the “The successful assembly of the asparagus YY genome…”\n- 143-149 and Mat & Methods: Somatic embryogenesis. There are few published papers that\ndescribes somatic embryogenesis using papaya hypocotyl, and genetic transformation and\nregeneration. I suggest that the authors describe whether their protocol is derived from one of\nthese articles and what improvements they have made to the present\nResponse: There are relatively few English publications on somatic embryogenesis in papaya,\nalthough some have been published in Chinese. Typically, papaya explants such as young leaves,\nimmature embryos, and hypocotyls around 10 days old are used for inducing embryogenic callus\n(Christopher, 2015). After testing various explants, we selected hypocotyls due to their high callus\nactivity and induction rates, although detailed formulations for embryogenic callus induction have\nnot been reported (Christopher, 2015).\nIn our study, we employed three different media for somatic embryogenesis in papaya. The\nconcentration of 2,4-D in the CMM medium was primarily based on the callus induction\nformulation by Zhu et al. Our significant improvement lies in the use of three distinct media—K5,\nM13, and CMM—each specifically tailored for explant differentiation, callus formation, and\nembryogenic callus induction, as opposed to the single-medium approach commonly found in the\nliterature. K5 and M13 media were independently developed and tested in our lab. This method\nnot only mitigates the variability in induction rates due to cultivar specificity but also reduces the\nembryogenic callus induction cycle by five months. This system has been publication (Zhao et al.,\n2021). In developing our papaya genetic transformation system, our laboratory adapted the\ntransformation method from Zhu et al. However, we optimized the formulation of the regeneration\nmedium by adjusting the concentrations of 6-BA and NAA in MBN medium, as well as the\nconcentration of IBA in the rooting medium. These modifications resulted in improved efficiency\nof both shoot regeneration and rooting (Zhu YJ, 2006). Establishing an efficient somatic\nembryogenesis and genetic transformation system for papaya is crucial for advancing gene\nfunction studies in this species. I have included the relevant references in the methods section.\nThe Mat & Methods we have added the references as follows:\n“........ The embryogenic callus induction system mainly refers to the protocol previously\nestablished in our laboratory, the three-step method for embryogenic callus induction significantly\nimproved induction efficiency and shortened the induction period (Zhao et al., 2021, Christopher\net al., 2015).”\n“........The genetic transformation protocol was based on previously published studies, but the\nhormone concentrations in the culture medium were optimized to enhance regeneration and\nrooting efficiency (Zhu et al., 2006).”\nReferences\npapaya cultivar ‘Kapoho’ from embryogenic cell suspension cultures. In Vitro\nCell.Dev.Biol. 2015, 580-587.\nZhao, X., Song, J., Zeng, Q., Ma, Y., Fang, H., Yang, L., Deng, B., Liu, J., Fang, J., Zuo, L.,\nand Yue, J. (2021). Auxin and cytokinin mediated regulation involved in vitro\norganogenesis of papaya. J Plant Physiol 260, 8.\nZhu YJ, F.M., Moore PH. (2006). Papaya (Carica papaya L.). In: Wang K (ed) Methods in\nMolecular Biology. (Totowa: Humana Press Inc.)\n- 152: “to producing” should read “to produce”\nResponse：Changed.\nLine 215: Which population does YYh comes from? Looking at the Hi-C heatmap data\n(Supplementary Fig. 21), it appears to come from genotype 15-4YYh. It would be helpful to\nindicate this information in the manuscript.\nResponse: We have indicated in the methods section that the assembly is 15-4 YYh, but this was\nnot mentioned in the main text. In response to one reviewer’s expert recommendations, we have\nrestructured the manuscript to improve its clarity and focus., and have removed the YYh genome\nsection to better highlight the primary objectives of the study.\n- 364-365: The sentence is unclear. Suggestion: “We focused on genes specifically expressed at\nthe 15-day stage between two samples.”\nResponse：Changed as “Our focus was on genes specifically expressed at the 15-day stage\nbetween female and hermaphrodite ovules. ”\n- 370-375: check the grammar\nResponse: The paragraph has been revised as follows:\n“Sucrose metabolism provide energy for embryonic development . The key enzymes\ninvolved in this process including alkaline/neutral invertases (A/N-Inv-C), which are localized in\nthe mitochondria, and catalyzes the breakdown of sucrose into glucose and fructose, thereby\nfueling various cellular processes. Additionally, a suite of other enzymes, including hexokinase 4\n(HXK4), cytoplasmic phosphoglucose isomerase (CPGI), PFK3, fructose-1,6-bisphosphate\naldolase (FBA6), fructokinase (FRK), triosephosphate isomerase (TPI), and fructose-bisphosphate\naldolase 6 (FBA6), play significant roles in this metabolic cascade. ”\n- 470: CpYYL was .... should Read CpYYL is ….\nResponse: “CpYYL was...” has changed as “CpYYL is an ANK repeat proteins with five ankyrin\n8\nrepeats, serving as an essential chloroplast protein for normal development of embryos in\nArabidopsis.” Line 365-366\n- Line 477-481: Something is missing in the sentence “Utilizing a complementation strategy\nwhere the emb506 and akrp mutants in Arabidopsis were partially complemented by\noverexpressing CpYYLand CpAKRP gene, respectively”. Please rewrite\nResponse: The sentence was changed as “Utilizing a complementation strategy, the emb506 and\nakrp mutants in Arabidopsis were partially complemented by the overexpression of the CpYYL\nand CpAKRP genes, respectively.” Line 372-374\n- Line 1108: The NCBI accessions are missing and are marked as (XX)\nResponse: We have completed uploading all the data, which is included under Data Availability as\nfollows:\n“Data availability: The raw Illumina RNA-seq data are publicly accessible in the Sequence Read\nArchive (SRA) under the BioProject accession number PRJNA1169527.”\n- Materiel & Method: The papaya cultivar used in paraffin sections (Figure1h,i ; Supplementary\nFigure2) is not described.\nResponse: Changed. The papaya cultivar used for paraffin sections and in situ hybridization was\nthe Zhongbai cultivar, the information has been added as follows:\n“For paraffin section analysis, seeds of Zhongbai papaya variety were collected at 35, 42, 49, 65,\n72, and 79 DAP and fixed in FAA (formaldehyde–acetic acid–ethanol) solution.” Line 808-810\n- Figure1 a,c: The photos show that the male flower has 5 stamens and the hermaphrodite has 10\nstamens, but the inverse is written in the figure legend?\nResponse: Changed. This was a writing error. Hermaphrodite plants have ten stamens and five\npistils, while male flowers have ten stamens. The figure legend has been corrected to: “a,\nHermaphrodite flowers showed short peduncles with ten stamens surrounding five pistils.”\n- Figure3a: The color code is not described. Is it the same as in “f”. Please precise in the. Figure\nand/or I the legend.\nResponse: The color code in Figure 4a, it is the same as in Figure 4f. Based on one reviewer’s\nprofessional suggestions,we have reorganized the entire manuscript to enhance its clarity and\nfocus, we removed the content of Y and Yh structural variations to clarify the main objective of the\npaper.\n9\n- Supplementary Figure19c: in the legend, I believe that “SunUp female and YYh20-1, 20-1XYh\n(15-4H) and 20-1XY (15-4M) were selected for further experiments” should read :\nSunUp female and YYh20-1, 20-1XYh (20-1H) and 20-1XY (20-1M) were selected for further\nexperiments”.\nResponse: This is an error. The label 15-4XYh (15-4H) and 15-4XY (15-4M) has been modified\nas 20-1XYh (20-1H) and 20-1XY (20-1M). Based on one reviewer’s professional suggestions,we\nhave reorganized the entire manuscript to enhance its clarity and focus, we removed the content\nrelated to the “YYh haplotype genome reveals structural variations between MSY and HSY” to\nclarify the main objective of the paper.\n- It will be helpful to people not in the papaya community, to mention in the beginning of the main\ntext, the diploid level and chromosome number of papaya.\nResponse: Thank you very much for your suggestion. We have added the following at the\nbeginning of the second paragraph of the introduction:\nPapaya is a diploid species with 9 chromosomes and a genome size of 352 Mb (Yue et al.,\n2022). Line 67.\nReference\nYue, J., VanBuren, R., Liu, J., Fang, J., Zhang, X., Liao, Z., Wai, C.M., Xu, X., Chen, S.,\nZhang, S., Ma, X., Ma, Y., Yu, H., Lin, J., Zhou, P., Huang, Y., Deng, B., Deng, F.,\nZhao, X., Yan, H., Fatima, M., Zerpa-Catanho, D., Lin, Z., Yang, M., Chen, N.J.,\nMora-Newcomer, E., Quesada-Rojas, P., Bogantes, A., Jiménez, V.M., Tang, H.,\nZhang, J., Wang, M.L., Paull, R.E., Yu, Q., and Ming, R. (2022). SunUp and Sunset\ngenomes revealed impact of particle bombardment mediated transformation and\ndomestication history in papaya. Nat Genet 54, 715-724.\n- Discussion section: I suggest that the authors discuss the link between CpYYL and sugar content\nand how CpYYL impacts sucrose metabolism?\nResponse: Thank you very much for your suggestion. We have added a section in the discussion to\nexplore how CpYYL influences sugar metabolism, as follows:\n“During early embryogenesis, proplastids depend on cytoplasm-generated hexose\nphosphates, like glucose-6-phosphate, for starch biosynthesis (Kammerer et al., 1998). These\nhexose phosphates also supply reducing power for plastid metabolic reactions through the\noxidative pentose phosphate pathway (Schwender et al., 2003). In Arabidopsis, lethal mutants\nemb506 and akrp have embryos arrested at the globular stage. Their papaya orthologs CpYYL and\nCpAKRP likely function similarly to play an essential role in plastid differentiation to regulate\nembryo development.\nHowever, the loss of CpYYL function may cause proplastid developmental defects in the\nearly embryo development of YhYh genotype, subsequently impairing the supply of energy and\ncarbon skeletons required for ovule development. This disruption of carbon flow balance likely\n10\npromotes the upregulation of CpInv-C and key glycolytic enzymes to rapidly generate energy\n(Kruger and von Schaewen, 2003). As a result, sucrose breakdown is accelerated (Fig. 6), a\nsignificant reduction in sucrose content was observed in the 15-day hermaphrodite ovules.\nExcessive activation of glycolysis in the cytoplasm depletes sucrose and glucose reserves, while\nproplastid developmental defects in YhYh embryos hinder the replenishment of carbon skeleton.\nTogether, these effects exacerbate cellular energy depletion and may ultimately lead to embryo\nabortion. These findings open a promising research avenue to further explore how CpYYL\nregulates the sucrose metabolic pathway and thereby influences embryo development. ”\nReferences\nKammerer, B., Fischer, K., Hilpert, B., Schubert, S., Gutensohn, M., Weber, A., and Flügge,\nU.I. (1998). Molecular characterization of a carbon transporter in plastids from\nheterotrophic tissues: the glucose 6-phosphate/phosphate antiporter. Plant Cell 10, 105-\n117.\nKruger, N.J., and von Schaewen, A. (2003). The oxidative pentose phosphate pathway: structure\nand organisation. Curr Opin Plant Biol 6, 236-246.\nSchwender, J., Ohlrogge, J.B., and Shachar-Hill, Y. (2003). A flux model of glycolysis and the\noxidative pentosephosphate pathway in developing Brassica napus embryos. J Biol Chem\n278, 29442-29453.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nThe manuscript \"Recreating viable YhYh genotype uncovers a domestication event of the Yh\nchromosome in papaya\" presents a groundbreaking study on the domestication and genetic basis\nof sex determination in papaya with a focus on the Y chromosome. Key findings include the\nidentification of the YY lethality gene (CpYYL) and its role in embryo viablity as well as the\ndiscovery of a retrotransposon insertion in the CpMp-h promoter as a crucial domestication event.\nMoreover, the authors successfully generated viable YhYh and YYh genotypes, addressing a long-\nstanding challenge in papaya breeding. Although papaya is not a classical easily manipulated lab\nmodel, the authors provide strong functional validation of the genes studied. The figures\neffectively illustrate key results, such as FISH validation, structural variants, and methylation\npatterns.\nHowever, the manuscript is dense particularly in the Results section. Reorganizing for better\nreadability — such as adding subheadings or including a summary figure of the experimental\nworkflow — would be helpful.\nmanuscript to improve its clarity and focus. As a result, we have repositioned the three sections on\nCpYYL to the beginning for improved logical coherence. we have also removed the CpMp gene\nsection and the 12 CpYYL transgenic insertion sites to better highlight the primary objective of the\npaper. Since each section already includes its own subheading, we have consolidated the research\ncontent on CpYYL into a schematic diagram. This diagram illustrates the molecular mechanism of\nYY lethality, the molecular function of CpYYL (Fig. 6).\nSome terms (e.g., \"stage 3 sex chromosomes\") are introduced without sufficient context for non-\nspecialists.\nResponse: We have rephrased the explanation for \"stage 3 sex chromosomes\" and added relevant\nbackground information as follow:\n“Based on current findings of various early stages of sex chromosomes, six stages of their\nevolution have been proposed. In stage 1, male sterility and female fertility genes are located close\ntogether on the same chromosome. In stage 2, recombination between these sex-determining genes\nis suppressed, leading to the formation of a small MSY, while the YY genotype remains viable, as\nseen in asparagus. By stage 3, recombination suppression extends to adjacent regions of the MSY,\nrendering the YY genotype lethal, as exemplified in papaya. The hallmark of stage 3 sex\nchromosomes is YY lethality (Ming et al., 2011) (Line 400-406).”\nReference\nMing, R., Bendahmane, A., and Renner, S.S. (2011). Sex chromosomes in land plants. Annu\nRev Plant Biol 62, 485-514.\nThe partial lethality of YhYh/YYh genotypes (59.6% and 26.9% mortality) is attributed to missing\nX-specific genes but lacks mechanistic detail. It would be beneficial to discuss whether this\nlethality results from haplo insufficiency or specific gene interactions.\nResponse: We observed that the partial lethality of YhYh/YYh genotypes, with mortality rates of\n59.6% and 26.9%, respectively, is primarily due to developmental defects that become apparent\nafter the cotyledon stage, leading to eventual plant death. These observations indicate that both\ngenotypes are indeed developmentally compromised. The most direct cause of this lethality may\nthe absence of X-specific genes in YhYh/YYh genotypes, which are present in XX and XYh\ngenotypes. We hypothesize that the Y chromosome carries a partially penetrant loss-of-function\nmutation in genes essential for early seedling development, which remain functional on the X\nchromosome. This hypothesis aligns with the theory of Y chromosome gene degeneration (Veltsos\net al., 2018; Veltsos and Delph, 2019)\nWe have added the following text to the discussion:\n“We hypothesize that the Y chromosome may carry a partially penetrant loss-of-function mutation\naffecting genes crucial for early seedling development, which remain functional in the X\n12\nchromosome. This hypothesis aligns with the theory of Y chromosome gene degeneration (Veltsos\net al., 2018; Veltsos and Delph, 2019). ”\nReferences\nVeltsos, P., and Delph, L.F. (2019). The X chromosome is necessary for ovule production in\nSilene latifolia. PLoS One 14.\nVeltsos, P., Cossard, G., Beaudoing, E., Beydon, G., Savova Bianchi, D., Roux, C., S, C.G.-\nM., and J, R.P. (2018). Size and Content of the Sex-Determining Region of the Y\nChromosome in Dioecious Mercurialis annua, a Plant with Homomorphic Sex\nChromosomes. Genes 9.\nFor improved clarity, adding a schematic model summarizing the molecular mechanism of YY\nlethality, CpYYL function, and the domestication of Yh is recommended.\nResponse: We have included a model in Figure 6 that summarizes the molecular mechanism\nbehind YY lethality and the role of CpYYL. Our diagram illustrates that the sterility of YhYh seeds\nin papaya is caused by the loss of function of the CpYYL gene on the Yh and Y chromosome,\nleading to the lethality of YY, YYh, YhYh homozygous embryos. In YhYh, the upregulation of key\nglycolytic enzymes and intensified sucrose metabolism were observed, exacerbating cellular\nenergy depletion in YhYh plants, which may lead to embryo sterility. According to another\nreviewer's suggestion, we have added a discussion explaining the link between CpYYL and sugar\ncontent, and how CpYYL impacts sucrose metabolism.\nDespite these comments, the manuscript represents a significant contribution to understanding the\nevolution and domestication of papaya, and I recommend it for acceptance after revisions.\nResponse: Thank you very much for recognizing our work.\n13"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The topic of the revised manuscript, entitled “Recreating viable YhYh genotype uncovers the role of CpYYL in papaya embryo development,” is focused on the gene (CpYYL) for YhYh (YY) lethality in papaya. I found its content to give a clear impression. For that reason, the function of the CpYYL gene and the traits and phenotypes of the developed YhYh plants must be impactful both scientifically and practically in the article in Nature Communications.\nIdentification of CpYYL as the causal gene for YY (YhYh) lethality and successful production o YhYh and YYh individuals are definitely novel and scientifically significant results. Honestly, I expected to be demonstrated that successful production of hermaphrodite papaya plants by crossing XX female and YhYh plants, which must be innovation of papaya cultivation. If such results are involved in the manuscript, it will be worth publishing without any doubt. But, according to the description in the discussion (p.9), results of crossing between female and YhYh was yet obtained.\nApart from this, still I have a few questions in the results to be cleared.\n- As commented in the previous review, I and the readers wish to know how identified CPYYL(CpEMB506) as the candidate gene for YY lethality. In the revision, more details are described, but additional informations are required. For example, predicted gene models were used for screening?, expression data was employed, and how many genes were observed?\n- It was described that HSY and MSY allele of CpYYL was the pseudogene in the text. According to the its structure, it could be expected. Did you analyzed its expression (transcripts) and confirm that its allele did not express?\n- Developed YhYh and YY plants seemed to show male phenotype in flower. Because it was quite important finding in this study, their sex phenotype should be described more apparently.\n- In the discussion, it was mentioned that completely CpYYL-lacked plants would be lethal. No its paralogous genes are present in papaya autosome? I suggest to describe whether CpYYL (CPEMB506) was unique or not in papaya genome.", "reviewer_2": "The authors have done an excellent job in responding to all comments. This revised manuscript is significantly improved, the data are convincing and open up new perspectives for papaya breeding.\nI have no further comments and recommend acceptance of this manuscript.", "reviewer_4": "I am completely satisfied with the response to my comments - in this form I have no further remarks on the manuscript."}, "author_response": "Reviewer #1 (Remarks to the Author):\nThe topic of the revised manuscript, entitled “Recreating viable YhYh genotype\nuncovers the role of CpYYL in papaya embryo development,” is focused on the gene\n(CpYYL) for YhYh (YY) lethality in papaya. I found its content to give a clear\nimpression. For that reason, the function of the CpYYL gene and the traits and\nphenotypes of the developed YhYh plants must be impactful both scientifically and\npractically in the article in Nature Communications.\nIdentification of CpYYL as the causal gene for YY (YhYh) lethality and successful\nproduction o YhYh and YYh individuals are definitely novel and scientifically\nsignificant results. Honestly, I expected to be demonstrated that successful production\nof hermaphrodite papaya plants by crossing XX female and YhYh plants, which must\nbe innovation of papaya cultivation. If such results are involved in the manuscript, it\nwill be worth publishing without any doubt. But, according to the description in the\ndiscussion (p.9), results of crossing between female and YhYh was yet obtained.\nResponse: We appreciate your constructive comments. In response to your questions,\nwe harvested seeds from plants initially marked as YhYh hermaphrodite and\ngenotyped the embryos. To our dismay, we detected XX female individuals, and\nfurther verification confirmed the three presumed YhYh hermaphrodites had been\nmisidentified and they were XYh hermaphrodite plants. After careful re-examining all\nYhYh DNA samples using redesigned markers followed by re-sequencing, we\nconfirmed all 18 true YhYh seedling died, indicating post-embryonic lethality. In\ncontrast, only 32 (25.4%) of the 126 YYh seedlings died at the seedling stage. This\ndifference in survival rate is likely due to a combined effect of different methylation\nbetween the MSY and HSY regions, and the absence of two Y-specific genes (CpMp\nand the unidentified carper suppressor) in YhYh genotype and 34 X-specific genes in\nthe YYh and YhYh genotypes.\nOur primary objective was to identify the gene controlling YY lethality, which we\nhave successfully achieved this goal. Obtaining viable YhYh hermaphrodite plants\nwould be an added benefit, as such plants could be directly utilized in papaya\nbreeding. We currently have multiple ProCpEMB506::CpEMB506/F1 XYh\nhermaphrodite plants in the field and will continue screening for viable YhYh\nseedlings, hoping that one may possesses a different methylation state that allow it to\ndevelop into an adult plant. However, there is no guarantee that such plants will be\nobtained.\nWe have been working on this project for 10 years, and achieving our primary\nobjective marks a major milestone. This manuscript presents the first identification\nand validation of a YY lethality gene in plants, and we hope the novelty and\nsignificance of these findings merit publication. We added two paragraphs to\nhighlights the importance and broader impact of our findings on sex chromosome\nevolution:\n1\n“Identification of the gene controlling YY lethality is just as challenging as\nidentification of the sex determination genes due to the large MSY that lacks\nrecombination, rendering forward genetic approach unfeasible. There is no precedent\nof identifying the gene controlling YY lethality and recreating viable YY genotypes.\nRecreation of viable YYh and YhYh seedlings through complementation test validated\nthe function of CpYYL that caused YY lethality when there is no functional HSY and\nMSY alleles. We recently identified the gene controlling the long male peduncles,\nCpMp, which play an essential role in the transition of papaya sex chromosomes from\nstage 1 to stage 2 (Zhou et al., 2025). The long male peduncles resulted in\napproximately 400 times more pollen production than hermaphrodite flowers,\nproviding a massive selective advantage that facilitated rapid elimination of\nhermaphrodite trees and firmly establish dioecy in the wild papaya populations (Zhou\net al., 2025). The identification of gene controlling YY lethality, CpYYL, reveals the\nmolecular mechanism underlining the transition of papaya sex chromosomes from\nstage 2 to stage 3. This transition eliminates the risk of viable YY supermale, which\nwould produce exclusively XY male offspring and ultimately drive population\nextinction.\nTo date, we have identified three of the five previously hypothesized factors in\nthe papaya SDR (Storey, 1953): CpMp, which controls long male peduncle; CpYYL,\nwhich is responsible for YY, YYh, and YhYh lethality; and two inversions within the\nSDR that suppress recombination (Wang et al., 2012). The two elusive sex\ndetermination genes remain to be discovered.”\nReferences\nStorey, W. (1953). Genetics of papaya. The Journal of Heredity 44, 70-78.\nWang, J., Na, J.K., Yu, Q., Gschwend, A.R., Han, J., Zeng, F., Aryal, R., Vanburen, R.,\nMurray, J.E., and Zhang, W. (2012). Sequencing papaya X and Yh chromosomes\nreveals molecular basis of incipient sex chromosome evolution. Proc Natl Acad Sci U S A\n109, 13710-13715.\nZhou, Y., Pang, Z., Wang, W., and Ming, R. (2025). Identification and molecular analysis of the\nY-specific CpMp gene controlling long male peduncles in papaya. Nat Commun 16,\n025-65632.\nApart from this, still I have a few questions in the results to be cleared.\n- As commented in the previous review, I and the readers wish to know how identified\nCPYYL(CpEMB506) as the candidate gene for YY lethality. In the revision, more\ndetails are described, but additional informations are required. For example, predicted\ngene models were used for screening?, expression data was employed, and how many\ngenes were observed?\nResponse: We revised the paragraph describing how we identified CpEMB506 as the\ncandidate gene for YY lethality:\n2\n“Given the lethal phenotype observed in YY/YhYh individuals, we hypothesize\nthat the gene responsible for YY/YhYh lethality remains functional in the X\nchromosome but becomes non-functional in the MSY and HSY. To investigate this,\nwe screened 34 X-specific genes 9 (Supplementary Table 1). Functional annotation\nidentified PYhCpXYh7 encodes an ankyrin repeat domain-containing protein\northologous to Arabidopsis EMB506, which is an essential gene for normal embryo\ndevelopment18. Transcriptomic analysis revealed CpEMB506 is highly expressed\nacross four embryonic stages, with its expression level gradually declining as\ndevelopment proceeds (Supplementary Table 1). The gene function and expression\npattern made CpEMB506 as a strong candidate gene underlying YY, YYh, and YhYh\nlethality.\nThe X allele features a large first intron of 8374 bp, which includes DNA\ntransposons and LTR retrotransposons. In contrast, the HSY allele has lost the first\nexon (409 bp) and 7352 bp in the first intron, rendering it a pseudogene (Fig. 2a and\nb). A comparison of the coding DNA sequences of X, HSY, and MSY alleles revealed\nthat the disrupted MSY and HSY alleles share conserved sequences, but differ from\nthe X allele by SNPs and Indels (Supplementary Fig. 3). Further quantitative PCR and\nRNA-seq data showed that the CpEMB506-X allele is expressed in both embryos and\nfemale flowers, whereas Cpemb506-HSY pseudogene is not expressed\n(Supplementary Fig. 4).”\nWe primarily focused on X-specific genes related to embryonic development and\nfurther identified candidate genes through expression data. Out of the 34 X-specific\ngenes, we selected one candidate gene, CpEMB506, because of its known function\nand the expression profiles of the X allele and HSY pseudogene.\n- It was described that HSY and MSY allele of CpYYL was the pseudogene in the text.\nAccording to the its structure, it could be expected. Did you analyzed its expression\n(transcripts) and confirm that its allele did not express?\nResponse: Yes, we performed expression, but we apologize for not providing the\nresult earlier. We designed quantitative primers specifically targeting the pseudogene\ngene Cpyyl-HSY to assess its expression, and the results showed no expression\n(Supplementary Fig. 4a). Additionally, visualization of the ovule transcriptome data\nfrom hybrid offspring of Zhongbai females × hermaphrodites crosses at 5d, 15d,\n35d,\nand 65d revealed strong read enrichment across the exons of the CpYYL-X, whereas\nCpyyl-HSY showed no read enrichment (Supplementary Fig. 4b). Together, these\nfindings demonstrate that Cpyyl-HSY is a non-functional pseudogene with no\nexpression.\n- Developed YhYh and YY plants seemed to show male phenotype in flower. Because\nit was quite important finding in this study, their sex phenotype should be described\nmore apparently.\ndevelopment arrests in YhYh plants. We have revised the description in the manuscript\nas follows:\n“The YYh genotype plants exhibited a male phenotype. Comparative analysis of\nof YYh and XY male plants and showed no significant differences in plant height,\nflower structure, and peduncle length compared to XY males (T- test, P = 0.14–0.97)\n(Fig. 2f-i and Supplementary Fig. 12). Scanning electron microscopy (SEM) confirmed\nthat pollen morphology and vitality were identical between YYh and XY male flowers\n(Supplementary Fig.13). ”\n- In the discussion, it was mentioned that completely CpYYL-lacked plants would be\nlethal. No its paralogous genes are present in papaya autosome? I suggest to describe\nwhether CpYYL (CpEMB506) was unique or not in papaya genome.\nResponse: We searched the annotated genes, and there is no paralog of CpYYL\n(CpEMB506). We have added the following text to our discussion:\n“The CpYYL X allele is an essential gene, and the Y or Yh pseudogene has no\nexpression. Moreover, there is no paralog of CpYYL on the autosomes. Therefore, any\nloss-of-function mutation in CpYYL would be lethal.”\nReviewer #2 (Remarks to the Author):\nThe authors have done an excellent job in responding to all comments. This revised\nmanuscript is significantly improved; the data are convincing and open up new\nperspectives for papaya breeding.\nI have no further comments and recommend acceptance of this manuscript.\nResponse: Thank you for your feedback.\nReviewer #4 (Remarks to the Author):\nI am completely satisfied with the response to my comments - in this form I have no\nfurther remarks on the manuscript.\nResponse: Thank you for your feedback.\n4"}, {"round": 3, "reviewer_comments": {"reviewer_1": "The revised version of the manuscript has been substantially improved and is suitable for publication."}, "author_response": ""}]

Decision letter