Going wild in banana breeding enables Fusarium-resistant hybrids with improved fruit quality

Domain: chemistry
Published: 2026-03-05
Editorial outcome: major_revision

Abstract
Bananas (Musa spp.) are crucial for food security, but modern cultivars derived from few wild species and propagated vegetatively possess low genetic diversity, and weak disease resistance. Identifying new parental lines is therefore essential for banana breeding. Here we show that Musa cheesmanii exhibits high resistance to Fusarium oxysporum f. sp. cubense tropical race 4. We cross it with two Musa ABB cultivars popular in China, 'Yulin' and 'Jinyu'. The resulting hybrids exhibit enhanced disease resistance, high yield, desirable pulp taste, and extended shelf-life, underscoring the breeding value of this wild species. Additionally, a telomere-to-telomere, gap-free genome of M. cheesmanii is assembled, with multi-omics analyses offering insights into its diversification, black pseudo-stem coloration, and disease resistance. Overall, M. cheesmanii is a promising male parent for enhancing genetic diversity and improving banana cultivars.

Introduction
Crop wild relatives (CWRs) are crucial for modern breeding programs as they provide essential genetic diversity that cultivated crops often lack1. These wild species carry crucial genes that can help overcome the genetic bottlenecks in cultivated crops, enhancing their resilience to climate change, pests, and diseases while improving yield and quality. Thus, protecting and utilizing the genetic diversity of CWRs is vital for the sustainability and adaptability of global agriculture.

Bananas (Musa spp.) are among the world's most important crops and are critical to food security in tropical and subtropical regions. However, global production is increasingly threatened by Fusarium wilt, a fungal disease caused by Fusarium oxysporum f. sp. cubense (Foc), with tropical race 4 (Foc_TR4) currently recognized as the most devastating fungal pathogen affecting banana cultivation worldwide2,3. Bananas' susceptibility to climate change, pest infestations, and diseases like Fusarium wilt highlights the urgent need for cultivar improvement to ensure sustainable production. The Musa genus, with approximately 70 wild species, is classified into two sections: sect. Musa (n=11) and sect. Callimusa (n=7/9/10)4,5. Most of the over 1,000 cultivated banana varieties originated from inter- and intra-specific hybridizations involving Musa acuminata (A genome), Musa balbisiana (B genome), and Musa schizocarpa (S genome)6-10. The majority of edible banana cultivars are found to be triploids (AAA, AAB and ABB), which have been selected by humans for their parthenocarpy11. Therefore, it appears that the genetic constitution of modern banana cultivars is derived from a very limited number of wild banana species with diverse hybridization history6,8,10,12. Most of the observed variation arises from variable mosaic contributions of different M. acuminata subspecies6,12. Additionally, the reliance on vegetative propagation (such as sucker division or tissue culture) in cultivated bananas has further narrowed genetic diversity, consequently weakened their resistance to numerous challenges3.

Hybrid breeding, which involves crossing two genetically distinct parents to combine their desirable traits, is a common approach for enhancing genetic diversity and improving crop characteristics. In the market, hybrid cultivars are generally more widely accepted than genetically modified varieties due to consumer preference for traditional breeding methods, which also serve as the primary approach for banana cultivar improvement. However, banana crossbreeding programs, which began nearly a century ago in Trinidad, continue to face significant challenges, such as the crop's triploid nature (resulting in low fertility) and lengthy breeding cycles. These challenges often result in breeding programs that take up to two decades to develop a locally acceptable cultivar, yet a commercially viable cultivar remains elusive13,14. Banana wild relatives are a promising source of genetic variation, offering a wide gene pool to enhance banana resilience against biotic and abiotic stresses. They typically produce numerous seeds and have large anthers with abundant pollen, making them ideal paternal resources in breeding programs. However, unlike major crops such as rice, wheat, and soybean, wild relatives have been used only to a very limited extent in banana breeding programs1. Traditionally, the diploid ancestors, subspecies of M. acuminata and M. balbisiana, are selected as the male parents.

M. acuminata comprises several subspecies, including banksii, malaccensis, zebrina, and burmannica. Among them, M. acuminata ssp. banksii is the major contributor to all cultivated bananas followed M. acuminata ssp. zebrina. Although M. acuminata ssp. burmannica, particularly the accession 'Calcutta 4', is not commonly found in natural cultivars, it has been widely used in breeding programs due to its strong disease resistance. Interestingly, malaccensis is also known for its high resistance to Foc, as well as a significant contributor to several cultivar groups, including AAA (e.g., Cavendish, Gros Michel, Red), AAB (e.g., Pome, Silk), and ABB (e.g., Pisang Awak)12. The use of M. balbisiana is severely constrained by the risk of activating endogenous banana streak virus (eBSV), especially under stress or during in vitro culture, which can lead to spontaneous viral expression in hybrids and compromise plant health and yield15-17. This genetic instability not only limits the use of B-genome cultivars in banana improvement programs but also hampers global germplasm exchanges due to the risk of eBSV activation into an infectious episomal form. Therefore, identifying potential male parents from banana wild relatives is promising for developing superior cultivars, given their significant role in enhancing cultivar diversity and their limited use in current breeding programs.

Species closely related to M. balbisiana (hereafter referred to as B-lineage species) have demonstrated high levels of resistance to Foc_TR418,19. Among the B-lineage species, Musa cheesmanii is phylogenetically close to M. balbisiana18. While it shares many morphological traits with M. balbisiana, M. cheesmanii can be distinguished by its significantly larger seeds, which are nearly twice the size, along with its more open bunch structure and upward-pointing fruits20. M. cheesmanii is a wild banana species native to Medog, China, the low-altitude southern slopes of the Himalayas, and the neighboring northeastern region of India20. It thrives in subtropical and tropical mountain forests, where it experiences cooler temperatures, higher humidity, and variable light levels compared to lowland species. This species features a tall (4 to 8 meters in height) and black pseudo-stem. The black appearance of the pseudo-stem is likely attributed to the accumulation of anthocyanins and/or the oxidative polymerization of phenolic compounds (e.g., catechols, L-DOPA, caffeic acid)21-23. According to our field observation, M. cheesmanii exhibits strong resistance to Fusarium wilt, as it remains disease-free when grown in soil contaminated with Foc_TR4; moreover. In addition, M. cheesmanii is highly productive, with its bunch weight exceeding that of most wild bananas by more than three-fold. Therefore, M. cheesmanii emerges as a highly promising candidate for use as a male parent in future banana breeding programs. Its use could significantly broaden the genetic base of bananas, leading to the development of high-yield, disease-resistant varieties. However, M. cheesmanii has not been systematically examined in banana breeding research, and its cross-compatibility and genetic characteristics remain unexplored, indicating the need for in-depth characterization to support its use in breeding.

In this study, offspring are generated by crossbreeding M. cheesmanii with two Musa ABB cultivars, 'Yulin' and 'Jinyu'. Cross-compatibility is assessed between three wild banana species, including M. acuminata, M. balbisiana, and M. cheesmanii, and the two ABB cultivars. Specifically, the ability of the wild species to serve as male parents in the crossbreeding process is evaluated. The results indicate that M. cheesmanii represents a promising male parent for banana breeding, as its hybrid offspring with ABB cultivars demonstrate enhanced Foc_TR4 resistance, high yield, favorable pulp taste, and extended shelf-life. A telomere-to-telomere (T2T) gap-free genome assembly of M. cheesmanii is also presented. Following this, we perform a multi-omics analysis to deepen genomic insights into the evolution of bananas, the molecular mechanisms underlying black pseudo-stem coloration, and the species' potential genetic variations related to disease resistance. Overall, M. cheesmanii is identified as a promising male parent for banana hybrid breeding, marking an advancement in the highly challenging field of banana breeding. Additionally, the comprehensive genome features of M. cheesmanii provide a foundation for future banana breeding efforts, paving the way for improved cultivars with enhanced traits.

Results
Response of M. cheesmanii and its offspring to Foc_TR4 infection
Fusarium wilt resistance was evaluated under both greenhouse and field conditions using Foc_TR4. The tested accessions included three wild bananas (M. acuminata ssp. malaccensis, M. balbisiana, and M. cheesmanii), two banana cultivars belonging to the ABB genome group ('Jinyu', Pisang Awak group, and 'Yulin', Dajiao group), their respective hybrids with M. cheesmanii ('Haifen No. 1' and 'Haijiao No. 1'), and a susceptible control, the Cavendish cultivar 'Baxijiao' (Table 1, Fig. 1). Several parameters were measured to evaluate Fusarium wilt resistance, including the incubation period (IP), which represents the average time from inoculation to symptom onset in greenhouse tests. Additionally, disease incidence was monitored by calculating the proportion of plants exhibiting external and internal Fusarium wilt symptoms out of the total number of plants evaluated. Disease severity and the disease index (DI) were also evaluated based on external symptoms and rhizome discoloration.

The IP of each accession under greenhouse conditions was shown in Table 1. The IP for the susceptible control 'Baxijiao' was the shortest among all the accessions tested, with symptoms appearing after an average of 34.3 days after inoculation (dai). M. balbisiana and M. acuminata ssp. malaccensis also exhibited Fusarium wilt symptoms, but their IPs were 3.5 and 10.8 days longer than that of 'Baxijiao', respectively. In contrast, M. cheesmanii showed no external symptoms, preventing us from calculating its IP. The IP of 'Jinyu' was 34.5 dai, comparable to that of 'Baxijiao', whereas its hybrid offspring 'Haifen No. 1' exhibited a longer IP of 38.3 dai. Similarly, 'Yulin' show a relatively long IP of 45.5 dai, while its hybrid 'Haijiao No. 1' displayed no external symptoms during the tested period. As expected, the 'Baxijiao' and 'Jinyu' showed the most severe symptoms of Fusarium wilt (Table 1, Fig. 1a, e), confirming their high susceptibility to Foc_TR4. The disease severity in the three wild relatives and the other cultivars was lower than in 'Baxijiao', yet Foc_TR4 still colonized these plants, causing characteristic Fusarium wilt symptoms, especially in M. acuminata ssp. malaccensis, M. balbisiana, and the hybrid 'Haifen No. 1' (Fig. 1b, c, f). Using the four-class rating scale developed in our previous study19, M. cheesmanii and the hybrid offspring 'Haijiao No. 1' were categorized as highly resistant, as no Fusarium wilt symptoms were observed externally, and only minimal rhizome discoloration was detected (Fig. 1d, h). Meanwhile, each hybrid demonstrated improved resistance compared to its corresponding maternal parent (Table 1, Fig. 1e-h).

In the field trials, symptoms of Fusarium wilt appeared in 'Baxijiao' and 'Jinyu' five months after planting. M. balbisiana and the hybrid 'Haifen No. 1' displayed noticeable yet mild external symptoms of Fusarium wilt during the dry and cold winter season of 2024. No external symptoms were observed in the other accessions throughout the duration of the field evaluation (Table 1). To confirm that Foc_TR4 was the causal agent of Fusarium wilt under field conditions, the nucleotide sequence of the pathogen was amplified from infected rhizome samples, as described in our previous study19. The results indicated that rhizome samples from 'Baxijiao', 'Jinyu', and M. balbisiana tested positive for Foc_TR4, those from 'Haifen No. 1' and M. acuminata ssp. malaccensis were occasionally positive, while samples from 'Yulin', 'Haijiao No. 1', and M. cheesmanii were generally negative.

Performance of hybrids of M. cheesmanii and Musa ABB cultivars
Crosses were made between two triploid cultivars ('Yulin' and 'Jinyu') and three diploid wild relatives (M. acuminata ssp. malaccensis, M. balbisiana, and M. cheesmanii), resulting in six unique combinations, which were evaluated for seed set (average number of seeds per fruit), seed germination percentage, offspring traits, and resistance to Foc_TR4 to assess their potential for banana breeding. The two triploid cultivars were female fertility identified in our previous study24. M. cheesmanii features a tall and black pseudo-stem (Fig. 2a), and crosses between 'Yulin'/'Jinyu' and M. cheesmanii displayed several outcomes (Table 2). Firstly, they showed the highest seed set (average number of seeds per fruit: 0.58±0.03). Secondly, the seed germination percentage was the lowest (≤ 1.7%), similar to that of the cross with M. acuminata ssp. malaccensis. Thirdly, the normal seedling (with healthy and regular appearance) percentage was moderate (slightly over 40%), being lower than that of the combinations with M. balbisiana but higher than those with M. acuminata ssp. malaccensis. Next, the offspring from these crosses exhibited the heaviest bunch weight among the three combinations with the same female parent; specifically, the offspring from the crosses between M. cheesmanii and 'Yulin' reached an average of 17.8 kg (significantly higher than the 10.0 kg and 13.5 kg observed with the other two male parents), and similarly, the offspring from the crosses between M. cheesmanii and 'Jinyu' showed an average bunch weight of 19.5 kg, outperforming the 13.5 kg and 16.1 kg recorded in the other combinations. In addition, the plant stature (defined as the ratio of plant girth to plant height) was the largest among the combinations with the same female parent; when 'Yulin' was used, the offspring from the cross with M. cheesmanii had a plant stature of 0.225, exceeding the 0.161 and 0.183 observed with M. acuminata ssp. malaccensis and M. balbisiana, respectively, while with 'Jinyu' the corresponding value was 0.171, slightly higher than the 0.167 and 0.163 seen with the other male parents, suggesting these plants may have relatively shorter, sturdier pseudo-stems that offer better resistance to wind damage and can support heavier bunches at maturity. Moreover, the majority of the offspring had red pseudo-stems, which were lighter than the deep black color of their male parent M. cheesmanii.

Significant heterobeltiosis was observed in the M. cheesmanii × 'Yulin' cross, with the hybrid showing complementary traits. First, it remained symptom-free when grown in Foc_TR4-infected fields (Table 1). Second, its bright red pseudo-stem provides additional ornament value (Fig. 2b,c). Third, its bunch weight was nearly double that of the maternal parent, with the hand arrangement resembling that of the paternal parent (Fig. 2d). Fourth, sensory evaluation showed that the hybrid fruit scored significantly higher than its maternal ABB cultivar for overall acceptability, indicating greater preference (Supplementary Table 1), while chemical composition analysis revealed significantly higher levels of β-carotene, soluble sugars, starch, carbohydrates, and sucrose (Supplementary Table 2), suggesting enhanced nutritional value and improved sweetness and overall eating quality. Fifth, it has an extended shelf-life of 6-10 days compared with 4-5 days for 'Yulin', with comparable fruit weight loss, slower softening of the pulp, and reduced malondialdehyde accumulation, supporting the observation that fruit quality remains largely unaffected despite peel blackening (Supplementary Fig. 1, Supplementary Table 3). Taken together, these results indicate that M. cheesmanii is cross compatible with certain cultivated bananas, yielding traits comparable to those of the two wild ancestors.

T2T gapless genome assembly and annotation of M. cheesmanii
The T2T gapless genome of M. cheesmanii was assembled by integrating PacBio high-fidelity (HiFi) reads, Oxford Nanopore Technology (ONT) ultra-long reads, and Hi-C reads (Supplementary Table 4, 5). The estimated genome size was 438.13 Mb, with a heterozygosity rate of 0.432% (Supplementary Fig. 2a). The assembled T2T gap-free genome is 432.16 Mb in size, consisting of 11 pseudo-chromosomes (Fig. 3a, Supplementary Table 6), and accounts for 98.63% of the estimated genome. Quality assessment showed that 99.52% of the HiFi reads mapped to the genome, indicating high coverage and assembly accuracy. Additionally, the LTR assembly index (LAI) reached 11.64, further supporting the reliability of the assembly (Supplementary Fig. 2b). Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis revealed 98.57% completeness for the 1,614 core conserved genes of embryophyta, whereas k-mer based analyses yielded a QV score of 48.97 and completeness of 99.00%, respectively, both supporting the high quality of the genome (Supplementary Table 7, Supplementary Fig. 3). Hi-C interaction data also provided strong evidence for accurate chromosome ordering and orientation (Supplementary Fig. 4). Genome segment comparison showed that nearly all segments of the two AAB cultivars (Plantain and Silk)25 could not be assigned to the M. cheesmanii genome (Supplementary Fig. 5), consistent with the fact that this species has not previously been utilized in banana breeding programs.

Repetitive sequences make up 46.65% of the genome, including 41.96% transposable elements (TEs) and 4.69% tandem repeats (Supplementary Data 1). Gene annotation predicted 38,197 genes (Supplementary Table 5). Functional annotation showed that 39.43%, 39.14%, and 69.37% of genes were annotated in the GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), and Pfam databases, respectively, resulting in 77.31% of genes being functionally annotated (Supplementary Table 8). Telomeric repeat analysis identified eleven pairs of telomeres with repeat lengths ranging from 1,081 to 5,376 base pairs (Supplementary Table 9). All eleven centromeric regions were identified based on TEs, tandem repeats, and Nanica LINE (Fig. 3a, Supplementary Table 10).

Genome evolution and demographic history of Musa species
Phylogenomic analysis of 9,388 single-copy orthogroups from M. cheesmanii, Ensete glaucum, and five other Musa species (M. acuminata, M. balbisiana, M. schizocarpa, M. troglodytarum, and M. beccarii) revealed that the Musa section splits into two major clades, A and B (Fig. 3b). In total, 34,302 orthogroups were characterized from 254, 531 genes (Supplementary Table 11), among which 7,683 gene families showed significant expansion or contraction (P < 0.05). In M. cheesmanii, 381 gene families were significantly expanded and 481 contracted. The most significantly enriched biological processes were related to carbohydrate metabolism, sugar transport, post-transcriptional processing, and ribosome biogenesis (Supplementary Data 2), while contracted genes were mainly involved in plant development, photoperiodism, and stress response (Supplementary Data 3). Gene family expansions in clade B were most significantly enriched in carbohydrate metabolism and defense mechanisms, while contractions mainly affected plant development pathways (Supplementary Data 4, 5). Genes involved in sugar transport and glucose import were significantly expanded in the Musa section but contracted in the Callimusa section, highlighting key evolutionary divergence (Supplementary Data 6-9).

Genome size variation was noted between the two sections, with Callimusa genomes ranging from 570 Mb to 603 Mb, and Musa genomes from 432 Mb to 525 Mb26,27,28,29,30. The M. cheesmanii genome is 432.16 Mb, with 46.65% repetitive sequences, similar in size and TE content to M. balbisiana26, but more compact compared to other Musa species (M. acuminata: 468.82 Mb, TE: 49.78%27; M. schizocarpa: 496.92 Mb, TE: 53.04%28; M. beccarii: 569.64 Mb, TE: 55.99%29; M. troglodytarum: 603 Mb, TE: 59.62%30). The smaller genome sizes of clade B species, including M. cheesmanii, are likely due to the lower abundance of intact LTRs, primarily from Copia and Gypsy families, in M. cheesmanii and M. balbisiana compared to M. acuminata and M. schizocarpa (Supplementary Fig. 6).

In M. cheesmanii, at least two whole-genome duplication (WGD) events were identified, consistent with previous findings in Musa species, with no species-specific duplications observed. Our analysis revealed 29,016 duplicated genes in M. cheesmanii, including WGD-derived, tandem, proximal, transposed, and dispersed duplication events. Tandem duplications (TD) were more common in the Musa section, while proximal duplications (PD) predominated in Callimusa species (Supplementary Fig. 7). The retained WGD expanded genes were mainly involved in housekeeping processes, while the PD and TD expanded genes were primarily associated with sugar transport, including glucose, hexose, monosaccharide, carbohydrate, and proton transmembrane transport (Supplementary Fig. 8, Supplementary Data 10, 11). These findings highlight the role of WGD in stabilizing housekeeping functions and maintaining genome stability, while TD and PD duplications may have facilitated metabolic innovations and species-specific adaptations in the wild Musa species.

Over the past one million years, the Earth experienced approximately nine glacial-interglacial cycles31. Demographic history analysis indicates that the effective population sizes (Ne) of all four studied species in the Musa section underwent an initial expansion, followed by a subsequent decline (Fig. 3c). However, clades A (M. acuminata and M. schizocarpa) and B (M. balbisiana and M. cheesmanii) exhibited distinct population trajectories, with clade A undergoing more pronounced Ne fluctuations during glacial-interglacial cycles compared to clade B. The demographic histories of the two clade A species are nearly identical, with Ne peaking around 200,000 to 400,000 years ago, followed by a sharp decline. In contrast, the two clade B species reached their Ne peaks earlier, approximately 500,000 to 780,000 years ago, and subsequently experienced a more gradual decline. Before the Last Glacial Maximum (LGM, approximately 20,000 years ago32), the Ne of these species reached a plateau. However, during the LGM, clade A experienced a sharp decline, while clade B remained relatively stable in Ne.

Chromosomal rearrangement and structural variation in Musa species
Extensive chromosomal rearrangements, including fusions, fissions, and translocations, were mainly identified in the Callimusa section, consistent with its variable chromosome numbers (Fig. 3b). Fusion of chromosomes 8 and 9 in the Musa section formed chromosome 8 in M. troglodytarum. In M. beccarii, more fusion and fissions events were observed. Between M. beccarii and M. troglodytarum, 135 syntenic blocks comprising 55,342 gene pairs were detected, with over half (77/135) scattered across different chromosomes (Fig. 3b). In contrast, the Musa section exhibited fewer rearrangements, though many occurred at hotpot regions overlapping with structural variation sites in the Callimusa section (e.g., chromosomes 1, 3, and 5). M. cheesmanii shared most conserved syntenic blocks with M. balbisiana and M. acuminata, except for a few translocations and inversions (Supplementary Fig. 9). No large chromosomal rearrangement was detected between M. schizocarpa and M. acuminata. Two major structural variations were identified in the Musa section: an inversion on chromosome 5 and a large reciprocal translocation between chromosomes 1 and 3 (Supplementary Fig. 9). Specifically, the chromosome 5 inversion was observed only in M. acuminata and M. schizocarpa, while the translocation between chromosomes 1 and 3 occurred between M. balbisiana and other Musa species studied. The read alignments around the breakpoints of these structural variations supported their existence (Supplementary Fig. 10).

Genome-wide survey of resistance genes
A genome-wide survey of R genes across M. cheesmanii, M. balbisiana, and M. acuminata was conducted, identifying 476 loci containing NB-ARC (Nucleotide-binding site) and LRR (Leucine-rich repeats) domains (NLR). Functional enrichments of these NLRs across the representative genomes consistently highlighted plant-microbe interactions, immune response, and defense-related pathways, supporting the robustness of R gene annotation (Supplementary Fig. 11). These NLR loci were classified into RESISTANCE TO POWDERY MILDEW 8-like coiled-coil NLRs (RNL), coiled-coil NLRs (CNL), and other NLRs33, whereas Toll/interleukin 1 receptor-like NLRs (TNL) were absent in banana. Among these categories, RNLs were the most abundant, accounting for 77.50%-83.64% of the total NLRs in each genome. Phylogenetic relationships of these NLRs and lineage-specific expansions and diversification events were presented in Fig. 4a. Orthogroup analysis revealed copy number variation of R genes between M. cheesmanii and M. balbisiana, as well as within M. acuminata (Fig. 4a, b). Some large translocations encompass multiple R gene clusters, such as the translocation involving chromosomes 1 and 3 between M. balbisiana and other Musa species, and the previously reported large reciprocal translocation involving 3 Mb and 10 Mb from chromosomes 1 and 4 within M. acuminata34 (Fig. 4c). In parallel, certain orthogroups, including OG0000000 and OG0000011, have shifted their chromosomal locations, reflecting similar patterns of genomic rearrangement (Fig. 4b). Moreover, many R genes were co-localized with transposable elements, suggesting a potential role of TEs in R gene family expansion and divergence (Supplementary Fig. 12).

Polymorphisms of eBSV integrants and RGA2
Banana streak virus (BSV), a pararetrovirus in the genus Badnavirus (Caulimoviridae), is approximately 7 kb in length with a double-stranded DNA genome comprising three consecutive open-reading frames (ORFs)35,36. To assess the suitability of M. cheesmanii as a male parent in banana hybrid breeding, endogenous BSV (eBSV) sequences were examined and compared with those in M. balbisiana. Initially, potential eBSV loci were investigated in the sect. Musa species, including M. acuminata, M. schizocarpa, M. balbisiana, and M. cheesmanii. No large viral sequences insertions were observed in M. acuminata and M. schizocarpa, with most blast hits fragment around 540 bp. In contrast, full-length BSV insertions were detected in M. balbisiana, such as two distinct BSV: banana streak IM virus (BSImV) and banana streak Mysore virus (BSMysV) sequences located on chromosome 2 (Supplementary Fig. 13a). However, M. cheesmanii harbored only large fragmental integrants, including three eBSV-like insetions regions: eBSV-like 1 on chromosome 5 (42,273,844-42,282,239 bp), eBSV-like 2 on chromosome 1 (6,376,588-6,396,669 bp), and eBSV-like 3 on chromosome 3 (15,045,005-15,056,027 bp). Phylogenetic analysis indicated their origin from a mixed subgroup containing isolate of BSV on a cultivar in Yunnan (BSAcYuV), a cultivar in Vietnam (BSAcVnV), and BSImV, which was distinct from M. balbisiana (Fig. 5a). Compared with the complete genome of BSAcYuV, the ORFs of the potential eBSV in M. cheesmanii were highly fragmented and rearranged (Fig. 5b-d). The eBSV-like 1 (Fig. 5b) and eBSV-like 3 (Fig. 5d) were relatively intact but lacked ORFI and had interrupted ORFIII due to stop codon insertions. Additionally, the viral integrants in M. cheesmanii diverged from the reference BSAcYuV with an overall similarity of around 80%, compared to more than 99% similarity to BSImV and BSMysV in M. balbisiana. Consistent with the genome sequences, the eBSV-like transcripts in M. cheesmanii were fragmented, whereas in M. balbisiana, these regions were generally expressed as complete transcripts (Supplementary Figs. 13b and 14).

It has been reported that introducing RGA2, a gene isolated from M. acuminata ssp. malaccensis, into Cavendish conferred resistance to Foc_TR4, with transgenic plants remaining disease-free over a three-year field trial37. This RGA2 gene consists of a single exon, and its ORF is 3,699 bp in length. To investigate its genetic variation, the ORF of RGA2 was compared across M. acuminata, M. balbisiana, M. cheesmanii, and 'Baxijiao'. In total, 166 single nucleotide polymorphisms (SNPs) were identified, approximately 57% of which were nonsynonymous mutations. A 6-bp insertion was identified at position 1,570 bp (site 1, Fig. 5e) in M. balbisiana and M. cheesmanii, potentially specific to B-lineage species. At 3,687 bp (site 2, Fig. 5e), a T-to-A mutation resulting in a premature stop codon was found in M. balbisiana and in two RGA2 homologs of 'Baxijiao' but not in M. cheesmanii. This mutation resulted in a truncated peptide chain with loss of six amino acids and subtle structural changes in the LRR domain, which is essential for pathogen recognition and signal modulation, altering β-sheet orientation and loop length (Supplementary Fig. 15). The nonsense mutation was confirmed by HiFi read alignments (Supplementary Fig. 16).

Transcriptomic and metabolic analyses reveal insights into pseudo-stem coloration in Musa species
To investigate the mechanisms underlying pseudo-stem coloration, comparative transcriptomic and metabolic analyses were conducted across Musa species: M. acuminata (green pseudo-stem, Mac), M. balbisiana (green pseudo-stem, Mba), and M. cheesmanii (green top pseudo-stem, Mch; black main pseudo-stem, Mchb). Principal component analysis (PCA) and transcriptional expression heatmap across species revealed significant transcriptomic differences, with good reproducibility among replicates (Supplementary Fig. 17a). While Mch and Mchb exhibited slight differences, differentially expressed genes (DEGs) analysis identified 7,098, 6,030, and 3,723 DEGs in comparisons of Mac vs. Mchb, Mba vs. Mchb, and Mch vs. Mchb, respectively (Supplementary Fig. 18). A total of 289, 314, and 338 genes in the phenylpropanoid (map00940), flavonoid (map00941), and anthocyanin biosynthesis (map00942) pathways were identified in M. cheesmanii, M. balbisiana, and M. acuminata, respectively (Supplementary Data 12-14). Besides, a total of 34 genes encoding polyphenol oxidase (PPO), peroxidase (POD), and DOPA decarboxylase (DDC) were identified (Fig. 6a). The expression patterns of these genes were then compared across the three species. Among these, 41 genes were up-regulated in Mch or Mchb (Supplementary Fig. 17b). Further pairwise comparisons of green (Mac, Mba, Mch) vs. black (Mchb) pseudo-stems identified 66, 50, and 44 DEGs. Only three genes were consistently up-regulated in Mchb and reflected the expression patterns observed across the three species: mch1s9g00634 (CCoAOMT), mch1s9g00701 (BGLU26), and mch1s2g02531 (PAL2) (Supplementary Fig. 19). Then additional comparisons based on mean-transformed expression were made between black and green pseudo-stems to focus on more anthocyanins-related structural genes (Supplementary Fig. 17b). Four flavonoid and anthocyanin biosynthesis genes, CHS (mch1s10g01364), FLS3 (mch1s10g02760), FE3'H (mch1s7g03161), and UFGT (mch1s10g02996), were up-regulated in the black pseudo-stem (Mchb) of M. cheesmanii across all replicate comparisons. We further performed quantitative RT-PCR (qPCR) to validate the expression of the seven identified genes in green and black pseudo-stems of M. cheesmanii. Two genes (BGLU26 and PAL2) failed to amplify, F3'H showed higher expression in black pseudo-stems but not significantly, while the remaining four genes were significantly up-regulated in black pseudo-stems (Supplementary Fig. 20). These four genes (CCoAOMT, CHS, FLS3, and UFGT) are therefore considered key candidates contributing to the black coloration of M. cheesmanii pseudo-stems (Fig. 6b). In addition, we performed qPCR analysis of F3'5'H, a key gene involved in the delphinidin-based pathway (Fig. 6a), and found that F3'5'H expression was elevated in the black pseudo-stem of M. cheesmanii (Supplementary Fig. 20). Among 3,498 identified transcription factors (TFs) in M. cheesmanii, 250 bHLH, 251 MYB/MYB-related, and 111 bZIP candidates were found (Supplementary Data 15-17). Further analysis identified 12 TFs consistently differentially expressed in Mchb, with 8 up-regulated and 4 down-regulated, including bHLH, MYB, and bZIP factors. Moreover, species-level comparisons (Mac vs. Mchb and Mba vs. Mchb) identified 6 transcription factors linked to light responses from a total of 37 identified, accounting for approximately 16% (Supplementary Fig. 21).

A total of 66 anthocyanin metabolites were identified across the three species, and the heatmap of hierarchical cluster analysis showed substantial differences (Supplementary Fig. 22). Among these metabolites, 3 belong to the pelargonidin-based pathway, while 27 and 29 are part of the cyanidin-based and delphinidin-based pathways, respectively. Most of these metabolites were much more abundant in M. acuminata (Supplementary Fig. 22), likely because it belongs to clade A and is more distantly related to M. cheesmanii. Therefore, we focused on comparing metabolite levels between black and green pseudo-stems of clade B species. We selected the top 10 most abundant metabolites in the black pseudo-stem (Mchb) of M. cheesmanii for comparison. Among them, six metabolites showed significantly higher abundances in Mchb than in the green pseudo-stems (Mch and Mba) (Fig. 6c). Two procyanidins (colorless or pale flavonoids) were the most abundant. Although they are known to polymerize into dark pigments through enzymes such as PPO or POD, transcriptome data revealed no significant up-regulation of PPO or POD genes in Mchb (Supplementary Fig. 19), suggesting that the role of procyanidins in pseudo-stem blackening is unclear. The remaining four metabolites were Delphinidin-3-O-rutinoside, Petunidin-3-O-rutinoside, Delphinidin-3-O-glucoside, and Petunidin-3-O-glucoside, all belonging to the delphinidin-based pathway, indicating that this pathway plays an important role in the black coloration of M. cheesmanii pseudo-stems.

Methods
Plant materials
Plant materials were collected from M. cheesmanii, which is conserved at South China Botanical Garden, Chinese Academy of Sciences, under the accession code ge-lz-013. The plant originated from South Himalaya (Medog, China), obtained in 2019. Currently, it is maintained in a greenhouse in Guangzhou, China, where it is actively monitored and utilized for ongoing breeding and genomic studies.

Fusarium wilt resistance evaluation
Eight accessions were selected for resistance testing. They included three wild banana relatives (M. acuminata ssp. malaccensis, M. balbisiana, and M. cheesmanii), two banana cultivars belonging to the ABB group ('Jinyu', Pisang Awak group, and 'Yulin', Dajiao group), their respective hybrids with M. cheesmanii ('Haifen No. 1' and 'Haijiao No. 1'), and a susceptible control, the Cavendish cultivar 'Baxijiao' (AAA). The M. cheesmanii accession was germinated from seeds collected in Medog, China, while the other seven accessions were propagated via tissue culture from plants grown at South China Botanical Garden or Guangxi Minzu University. Plantlets were grown in pots containing disinfected Tref substrates (Jiffy, Moerdijk, Netherlands). During the acclimatization period, the plants were watered daily and weekly supplemented with 50 mL per plant of Murashige and Skoog’s solution to promote healthy growth.

Greenhouse evaluations were carried out in 2024 under controlled conditions, with temperatures ranging from 26-35 °C and approximately 85% relative humidity. The strain used for testing was Fusarium oxysporum f. sp. cubense tropical race 4 (Foc_TR4, VCG01216). Briefly, for each accession, twenty-one plantlets with similar morphological characteristics (6-7 leaves and 30-50 cm in height) were selected. Fifteen plantlets were individually inoculated by pouring aliquots (2 mL per aliquot) of spore suspension (5 × 106 spores mL-1) directly into five equidistant holes around the plant base near the rhizome; the others treated with sterile water were kept as controls. The whole experiment was replicated three times.

Field evaluations were conducted at an experimental plot in Nanning, Guangxi province (22°48’ N, 108°22’ E; Alt. 106 m), which was naturally infested with Foc_TR4, from 2023 to 2025 (including three replicates). Briefly, fifteen acclimatized plants (40-50 cm in height) of each accession were planted in a randomized block design. Plants were supplied monthly with 50-150 mL of Murashige and Skoog solution per plant, depending on their developmental stage, and were watered weekly.

Fusarium wilt resistance was evaluated using several parameters, starting with the incubation period (IP), which refers to the average time (in days after inoculation, dai) between inoculation and the appearance of symptoms in greenhouse conditions. Disease incidence was measured by calculating the proportion of plants showing both external and internal Fusarium wilt symptoms relative to the total number of plants assessed. In addition, disease severity was evaluated and the disease index (DI) was determined by examining external symptoms and rhizome discoloration (internal symptom) according to Li et al.19. Briefly, disease severity of both external and internal symptoms were deemed according to the respective scale of 0-3. The DI was calculated as:
∑(score in the scale × frequency)
𝐷𝐼 = × 100 (1)
total number of plants × maximum class in the scale
In greenhouse, IP was assessed by visual inspection of initial external appearance of symptoms every 2 days starting 7 dai; internal symptoms were evaluated visually when a plantlet died or at the end of the experiments (65 dai). In field, evaluations of external disease intensity (incidence and severity) were performed every 2 weeks; when the plant showed serious symptom or at the end of the experiment (21 months after planting), internal symptoms (rhizome discoloration) were evaluated.

Cross-compatibility evaluation
Crosses were made between two triploid cultivars (female parents) and three diploid wild relatives (male parents), resulting in six unique combinations (Table 2). The two female parents used in the crosses were 'Yulin' (Musa ABB Dajiao group) and 'Jinyu' (Musa ABB Pisang Awak group). The Dajiao group is favored in South China, whereas the Pisang Awak group is widely popular in Southeast Asia and ranks as the second most consumed banana variety in China after Cavendish. Both cultivars are known for their low fertility. 'Yulin' and 'Jinyu', the representative cultivars of these two groups, were both grown at Guangxi Minzu University. The three male parents, M. acuminata ssp. malaccensis, M. balbisiana, and M. cheesmanii, were the same as those used in the Fusarium wilt resistance evaluation section. Each female parent was artificially crossed with each of the three male parents, resulting in a total of six crosses.

Artificial crossings were carried out continuously from 2022 onward. For each cross, pollen grains were carefully harvested from the male parent and transferred onto the receptive flowers of the female parent in the field. To prevent any accidental contamination from foreign pollen, the emerging female flowers and male pollen sources were carefully bagged. After the seeds were harvested, they were germinated in vitro to develop an F1 progeny population for each of the six combinations. The seedlings were then acclimated in a controlled greenhouse environment before being transplanted into a field naturally infested with Foc_TR4 to assess their resistance.

The seed set (average number of seeds per fruit), seed germination percentage, and several key morphological traits of the offspring, including normal seedling (with healthy and regular appearance) rate, bunch weight, pseudo-stem height, plant stature (defined as pseudo-stem girth at 150 cm above ground to the total height of the pseudo-stem when flowering), and pseudo-stem color, were evaluated to assess the cross-compatibility of the different combinations. Seed set, germination, and seedling architecture were evaluated using >3000 fruits, >300 seeds, and >80 progenies (each with three replicates). Pseudo-stem color was visually assessed in >100 plants, while bunch weight, pseudo-stem height, and plant stature were measured from ten parental plants and ten selected hybrid lines (five plants per line). These traits were critical for understanding the potential of each cross to contribute to banana breeding efforts, particularly in terms of disease resistance and other agronomically important characteristics.

To evaluate heterobeltiosis in agronomic traits, fruit quality was assessed for 'Yulin' and its hybrid 'Haijiao No. 1' ('Yulin' × M. cheesmanii). The evaluation included sensory assessment, chemical composition analysis, and shelf-life determination. For sensory evaluation, blind tests were conducted. Twenty-six panelists without oral diseases tasted fully ripe fruits from both cultivars and independently scored them on eight parameters: sweetness, acidity, banana aroma, texture, astringency, off-flavor, overall flavor, and overall acceptability. For chemical composition analysis, the ripe pulps of each cultivar were analyzed for sixteen parameters following the corresponding GB/T (National Recommended Standard of the People’s Republic of China) or NY/T (National Standard of Agricultural Industry of the People’s Republic of China) methods (Supplementary Table 2). For shelf-life assessment, fruits were stored at room temperatures, specifically from 32 °C at day to 24 °C at night. Shelf-life was monitored every two days by photographic documentation, sensory evaluation, and postharvest quality (including fruit weight loss, pulp firmness, and malondialdehyde content), starting from the end of the green-life stage (when the peel begins to yellow) until the end of the edible period. Weight loss rate was calculated as:
Weight on 0 day−Weight on checked day
𝑊𝑒𝑖𝑔ℎ𝑡 𝑙𝑜𝑠𝑠 𝑟𝑎𝑡𝑒 = × 100% (2)
Weight on 0 day
Pulp firmness and malondialdehyde content were tested following NY/T 2009-2011 and GB5009.181-216, respectively. For each cultivar, three biological replicates were used, each consisting of three fruits.

Genome sequencing
Fresh young leaves of M. cheesmanii were collected, immediately stored in liquid nitrogen, and then sent to BGI (Shenzhen, China) for genome sequencing. The CTAB method49 was used to isolate the total genomic DNA from the leaves. For PE150 (paired-end 150 bp) sequencing, libraries with an insertion size of 350 bp were constructed and sequenced on a DNBseq platform. The raw PE150 reads were then filtered using Fastp v0.23.250 with default parameters, generating 47.13 Gb of clean PE150 reads (Supplementary Table 4). PacBio HiFi sequencing was carried out on a Sequel II platform, yielding 38.39 Gb of clean HiFi reads after filtering with Fastp using the parameters: (-q 20 -l 1000 -A) (Supplementary Table 4). ONT sequencing was carried out on a PromethION platform. After filtration with Fastp with the parameters: (-q 7 -u 100 -e 7 -A -l 40000), 114.43 Gb of ONT clean reads longer than 40 kb were generated (Supplementary Table 4). Hi-C libraries were prepared by formaldehyde cross-linking, DpnII digestion, biotin labeling, proximity ligation, purification, and size selection, followed by sequencing on the DNBseq platform with PE150 reads. Finally, 139.81 Gb of Hi-C clean reads were produced after filtration using Fastp (Supplementary Table 4). For RNA sequencing (RNA-seq), total RNA from leaves and roots was extracted and sequenced on the DNBseq platform in the PE150 pattern. A total of 14.44 Gb of clean RNA reads were obtained after filtration using Fastp (Supplementary Table 4).

Genome survey and high-quality genome assembly
Based on HiFi reads, Jellyfish v2.3.051 was utilized to calculate k-mers of 17, 21, and 31, and the outputs were subjected to GenomeScope252 to assess genome size and heterozygosity. Hifiasm v0.19.353 was applied to assemble the HiFi reads with the parameter (--ul) to integrate the ONT reads. The generated preliminary assembled heterozygous contigs (p_ctg) were subjected to two rounds of polishing using NextPolish v1.4.154 with HiFi reads. Redundant contigs caused by heterozygosity were then eliminated using Purge_Haplotigs v1.1.255. Subsequently, the Hi-C clean reads were aligned to the purged genome assembly using BWA v0.7.1756, followed by Juicer v1.657 to identify valid interaction pairs. Finally, 3D-DNA v20100858 was used to anchor the final contigs into pseudo-chromosomes, and Juicebox Assembly Tools v1.11.0859 was used to manually improve the accuracy of the chromosomal assembly. Then, HiCExplorer v3.060 was used to visualize the Hi-C data.

To further improve genome quality, TGS-GapCloser v1.2.161 was applied to fill the remaining four gaps in the pseudo-chromosomes using the assembled ONT contigs generated from NextDenovo v2.5.0 (https://github.com/Nextomics/NextDenovo). The reliability of gap-filling sequences was validated by examing breakpoints using long-read alignments and Hi-C contact data (Supplementary Figs 23, 24). With each terminal 100 kb sequences as probes, minimap2 v2.2462 was performed against polished HiFi primary contig database for telomere repairing. Finally, a T2T gapless genome was generated after polished by NextPolish with HiFi reads. The mapping rate of final assembly was evaluated using SAMtools v1.1463. To monitor quality changes across different assembly stages, BUSCO v5.4.364, Merqury v1.365, and KAT v2.4.266 were employed for genome quality assessment. Overall, although polishing appeared to slightly lower the quality of the assembly, the results demonstrated consistently high-quality assemblies at every stage (Supplementary Table 7). The R package Rideogram67 was used to visualize and map genome-wide data on the ideograms based on 1-Mb windows.

To assess the potential contribution of M. cheesmanii to banana cultivars, we analyzed Plantain and Silk (AAB cultivars)25 using chromosome painting68 with multiple ancestral genome assemblies, including five M. acuminata subspecies (malaccensis, banksii, zebrina, burmannica, and halabanensis)9,27, M. schizocarpa28, M. balbisiana26, and M. cheesmanii, for segment comparisons.

Genome assembly annotation
Telomeric and centromeric regions were identified using quarTeT69. Using the repeat monomer AAATCCC/TTTAGGG, TeloExplorer (Module #3) was performed to identify telomeric regions, and TIDK v0.2.1 (https://github.com/tolkit/telomeric-identifier) was used to explore and search telomeric regions through automatic workflows. CentroMiner (Module #4) was utilized to predict centromere candidates using transposable element annotation in GFF3 format and a genome FASTA file as input. Based on period and copy number, centromeric tandem repeat monomers were screened, clustered to reduce redundancy, and aligned to corresponding chromosomes (Supplementary Table 10). The Nanica LINE reported in M. acuminata70 was downloaded (https://banana-genome-hub.southgreen.fr/node/50/353) as a probe for further checking the centromeric region in the M. cheesmanii genome, and blastn v2.11.071 was applied to perform searches.

Extensive de novo TE Annotator (EDTA) v2.1.072 was employed to discover TEs with the parameters: (--species others --step all --evaluate 1 --sensitive 1 --anno 1). The non-redundant TE library yielded by EDTA was then imported into RepeatMasker v4.1.073 to soft-mask repeat sequences in the genome using the parameter “-xsmall”. In addition, Tandem Repeats Finder v4.0974 was employed to identify tandem repeats. For comparative analysis of TE contents, the EDTA pipeline with identical parameters was used to annotate other Musa species. LTR_retriever v2.9.075 was used to extract intact LTR retrotransposons and calculate their insertion time within Musa species using a mutation rate of 1.3e-8 from the monocot species rice.

The standard MAKER3 v3.01.033876 pipeline was utilized to annotate gene structures. For homology prediction, all high-confidence protein sequences were imported from the Swiss-Prot database77, and raw gene structures were predicted based on tblastn alignments. The RNA-seq data were mapped to the T2T genome using HISAT2 v2.2.178, and genome-guided transcripts were obtained by StringTie v2.1.779. Then, AUGUSTUS v3.3.2 and SNAP v20131129 were employed to train the best predicted gene models. The MAKER3 pipeline was run twice to obtain more complete gene annotations. Finally, eggNOG mapper80 was used to functionally annotate the predicted coding genes based on the eggNOG v5.0 database81.

Comparative phylogenomics
Orthologous gene families were clustered using the longest transcripts from M. cheesmanii and six other Musaceae species, downloaded from the Banana Genome Hub (https://banana-genome-hub.southgreen.fr/)26,27,28,29,30,82,83. The remaining six species include three from the sect. Musa (M. acuminata, M. schizocarpa and M. balbisiana), two from the sect. Callimusa (M. troglodytarum and M. beccarii), and the outgroup, Ensete glaucum. Orthofinder v2.5.484 was employed to classify the gene families. Orthologous sequences were aligned using MAFFT v7.51585, trimmed using trimAl v1.2rev5986, and then concatenated. Phylogenetic tree was constructed using RAxML-NG v1.187. FigTree v1.4.4 (https://github.com/rambaut/figtree) was used to visualize the phylogenetic tree. MCScan88 was utilized to identify structural variations with the parameters: (--cscore .99, --minspan 30). Then, CAFE v589 was utilized to simulate the dynamics of orthologous gene family expansion and contraction. TBtools v2.04290 was used to perform GO enrichment analysis for significantly expanded and contracted gene families in M. cheesmanii. Hypergeometric tests combined with and the Benjamini–Hochberg (BH) method were applied to obtain adjusted P values, retaining only GO terms with a BH adjustment P < 0.05. The GO enrichment results were visualized using ChiPlot (https://www.chiplot.online/).

Whole-genome duplication and structure variation
WGDI91 was introduced to identify whole-genome duplications (WGDs) in M. cheesmanii and six other Musaceae species26,27,28,29,30,82. Protein sequences were subjected to all-against-all and inter-specific comparisons using BLASTP v2.13.0 in the BLAST package71 with the parameters: (-outfmt 6 -evalue 1e-5 -num_alignments 20). MCScan88 was employed to establish collinearity between gene pairs, filtering out non-collinear gene pairs and tandem duplicated genes. Then, MUSCLE92 was used to align the proteins. The MCScan python version was used to identify syntenic blocks among these Musaceae assemblies with the parameters: (--cscore .99, --minspan 30). DupGen-finder93 was used to identify various types of duplicated genes in these Musaceae species including WGDs, tandem duplications (TDs), proximal duplications (PDs), transposed duplications (TRDs), and dispersed duplications (DSDs). GEO enrichment analysis was conducted using TBtools v2.04290 on 583 expanded genes derived from TD and PD events. Significant GO terms were visualized using ChiPlot (https://www.chiplot.online/).

Estimation of demographic history in the Musa section
Paired-end reads of three Musa A-clade/B-clade species26,27,28, along with M. cheesmanii sequenced in this study, were mapped to the assembled genome using the BWA-MEM algorithm56. SAMtools v1.1063 was used to remove duplicate and unpaired error-mapped reads from the alignments. Then, mpileup was performed in BCFtools94 with the parameters: (-Ou -I), and variant calling was conducted using BCFtools with the parameters: (-c -Ov). The vcf2fq in vcfutils.pl with the parameters (-d 10 -D 100) was used to convert the consensus genotype from VCF to FASTQ format. Next, the evolutionary effective population size of the four sect. Musa species was inferred using the Pairwise Sequentially Markovian Coalescent (PSMC, https://github.com/lh3/psmc) model with the parameters: (-N25 -t15 -r5 -p ‘4+25*2+4+6’ ). The PSMC results were scaled using a generation time of 1 year and a mutation rate of 1.3e-8.

Resistance gene analysis
Resistance (R) genes across M. cheesmanii, M. balbisiana, and M. acuminata (MAv4, MAH1, MAH2) were identified using the FindPlantNLRs pipeline v2.033, which predicts loci containing NB-ARC and LRR domains from unmasked genome assemblies. To remove false positives, both the prediction and annotation stages were integrated. Orthogroups of R genes were inferred based on protein sequence similarity using OrthoFinder v2.5.484. Multiple sequence alignments were generated with MAFFT v7.52685 under default parameters and trimmed with trimAI v1.5.rev086 under -automated1 mode to remove non-conserved sites. Maximum likelihood phylogenetic inference was conducted using IQ-TREE v 2.4.095 with the best-fit model JTT+F+R6 and 1000 bootstrap. Phylogenetic trees were visualized with iTOL v796. GO enrichment analysis for R genes in each species was performed using TBtools v2.04290 (Significant at BH adjustment P < 0.05 level).

eBSV integrants
Available reference banana streak virus (BSV) and endogenous BSV (eBSV) sequences (DQ092436.1, KJ013508.1, AY750155.1, AY493509.1, ENC_003381.1, NC_006955.1) were downloaded from GenBank or NCBI database70,97,98. BLAST analysis for M. acuminata, M. schizocarpa, M. balbisiana, and M. cheesmanii was performed using full-length of BSV and eBSV or partial cds fragment of the ORFIII like region as described by D'Hont et al.70. Alignment regions with <500 bp length and <75% identity percentage were filtered. Then, in order to check whether complete eBSV integration exists, consecutive loci with alignment length exceeding 1,000 bp were focused on. Sequences were further extracted from the 2 kb upstream of the first alignment locus and the 2 kb downstream of the last one. Manual annotation and sequence codon frequency statistics were performed using Geneious Prime 2022.2.2 (https://www.geneious.com) with interior ORFs included and a 300 bp minimum size for finding ORFs. Reorganization with duplications and inversions was checked again by BLAST with E-value set as 1e-5 and visualized by TBtools90. Only BSV sequences with full-length and extracted consecutive regions were used to obtain a more reliable BSV phylogeny. All nucleotide sequences were aligned using MAFFT85 and then alignments were trimmed using trimAl86. Phylogenetic tree was constructed using IQ-TREE v2.3.695 with best-fit model TPM2u+F+R3 and 100 bootstraps.

RGA2 polymorphisms
RGA2 sequence was downloaded from GenBank (accession numbers EU616673)99. BLAST analysis for cultivar 'Baxijiao', M. acuminata, M. balbisiana, and M. cheesmanii was performed using RGA2 sequence as reference. The 1 kb upstream and downstream regions of the hit loci were extracted and examined for annotation and correction. All orthologous RGA2 sequences were aligned using ClustalW algorithm for DNA sequences and MUSCLE algorithm for protein sequences in MEGA11100. AlphaFold3101 was used to predict protein structures.

Transcriptome analysis of pseudo-stem in the Musa genus
To investigate why M. cheesmanii exhibits black color in its pseudo-stem, transcriptome analysis was performed on pseudo-stem samples from four groups (three replicates each): M. acuminata (Mac), M. balbisiana (Mba), and M. cheesmanii (group Mch/Mchb). For M. cheesmanii, samples were collected from the top pseudo-stem (green, Mch) and the main pseudo-stem (black, Mchb). For the other two species, samples were collected from the main pseudo-stem (green color). Total RNAs extracted from these pseudo-stems were sequenced on the DNBseq platform in the PE150 pattern. Subsequently, raw reads were trimmed and filtered using Fastp v0.23.250. Clean RNA-seq reads were then mapped to the assembled M. cheesmanii genome using HISAT2 v2.2.178, and raw counts were quantified using featureCounts v2.0 .4102. Differentially expressed genes (DEGs) between any two groups were identified using DESeq2103 with criteria set as |log2FoldChange| ≥ 1.0 and adjusted P ≤ 0.05. Besides, normalization expression level was calculated by transcripts per million (TPM) for convenient direct comparisons of all sampled groups. Based on transcriptomic data, functional annotation, and KEGG pathways (http://www.kegg.jp/kegg/pathway.html), candidate genes involved in phenylpropanoid biosynthesis (map00940), flavonoid biosynthesis (map00941), and anthocyanin biosynthesis (map00942) were identified in M. cheesmanii, M. balbisiana, and M. acuminata. Plant transcription factors (TFs), transcriptional regulators (TRs), and protein kinases (PKs) were identified using the iTAK program104. MYB, bHLH, and bZIP type key families of TFs, known for their roles in regulating anthocyanin biosynthesis, were identified.

Quantitative RT-PCR
For the qPCR quantification of pigmentation-related genes, the total RNAs were extracted from the green and black pseudo-stems of M. cheesmanii (Mch and Mchb). The extracted RNAs were reverse-transcribed into cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) according to the manufacturer’s protocol, and the resulting cDNA was subsequently diluted with 100 μL of RNase-free ddH2O. The specific primers used for amplification were synthesized using standard phosphoramidite chemistry and purified by polyacrylamide gel electrophoresis (Wuhan Tianyi Huayu Gene Technology Co., Ltd, China), and the sequences were listed in Supplementary Table 12. All data were collected from three biological replicates. The reaction system (20 μL) contained ChamQ Blue Universal SYBR qPCR Master Mix, 10 μL; 10 μM Primer 1, 0.4 μL; 10 μM Primer 2, 0.4 μL; cDNA, 2.2 μL; ddH2O, 7 μL. PCR cycles were 95 °C 5 m; 95 °C 10 s, 60 °C 30 s, 40 cycles. Relative mRNA level for each gene was calculated by 2-ΔΔCT methods with L2 set as reference gene105,106.

Anthocyanin metabolomic analysis
The pseudo-stem samples from M. acuminata (Mac), M. balbisiana (Mba), and M. cheesmanii (Mch/Mchb) were randomly collected in the South China Botanical Garden, Chinese Academy of Sciences (three biological replicates for each group, total n = 12). The samples were immediately frozen in liquid nitrogen, ground to powder (50 mg for each replicate) followed by freeze-dried, and stored at -80 °C. Subsequently, the dried tissues were extracted twice using 0.5 mL methanol/water/hydrochloric acid (500:500:1, v/v/v), followed by vortexing (5 min), ultrasonication (5 min), and centrifugation (12,000 g, 4 °C, 3 min). Supernatants were pooled and filtered through 0.22 μm membranes for LC-MS/MS analysis.

The sample extracts were analyzed using an UPLC-ESI-MS/MS system (UPLC, ExionLCTM AD, http://sciex.com.cn/; MS, Applied Biosystems 6500 Triple Quadrupole, https://sciex.com.cn/). Chromatographic separation was performed on a Waters ACQUITY BEH C18 column (2.1 × 100 mm, 1.7 μm) using a UPLC system with mobile phase A consisting of water (0.1% formic acid) and mobile phase B consisting of methanol (0.1% formic acid). The gradient elution program was set as follows: 95:5 V/V at 0 min, 50:50 V/V at 6 min, 5:95 V/V at 12 min, held for 2 min, 95:5 V/V at 14 min, held for 2 min. The flow rate was at 0.35 mL/min, the column temperature was maintained at 40 °C, and the injection volume was 2 μL.

Detection was performed on a QTRAP® 6500+ LC-MS/MS system in positive ESI mode with scheduled multiple reaction monitoring (MRM). The ESI source parameters were set as follows: ion spray voltage, 5500 V; source temperature, 550 °C; curtain gas, 35 psi. For each analyte, declustering potential (DP) and collision energy (CE) were individually optimized, and a compound-specific set of MRM transitions was monitored within defined retention-time windows according to chromatographic elution.

Metabolite quantification was performed using a triple quadrupole mass spectrometer (QTRAP® 6500+) in Multiple Reaction Monitoring (MRM) mode. In MRM, the first quadrupole (Q1) selects the precursor ion of the target metabolite, filtering out non-target ions to reduce interference. The precursor ion is fragmented in the collision cell (Q2), and the third quadrupole (Q3) selectively monitors a characteristic fragment ion, improving quantitative accuracy and reproducibility. Chromatographic peak areas of target metabolites were integrated for all samples and converted to concentrations using external calibration curves. Standard stock solutions (1 mg/mL in 50% MeOH) were diluted to working concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000, 2000, and 5000 ng/mL. Peak areas of standards were plotted against concentrations to generate calibration curves, which were used to calculate metabolite concentrations in samples. The final metabolite content (μg/g) in each sample was calculated as:
Metabolite content = c × V × 10−6 (3)
m
where c is the concentration derived from the calibration curve (ng/mL), V is the extraction volume (μL), and m is the sample mass (g).

Mass spectrometry data were processed using Analyst v1.6.3 (SCIEX, Framingham, MA), and peak integration and quantification were carried out using MultiQuant v3.0.3 (SCIEX, Framingham, MA). Total ion chromatograms (TIC) and extracted ion chromatograms (XIC) were used to visualize metabolite detection profiles and verify peak quality. Authentic standards were used when available, whereas compounds lacking purified standards were semi-quantified using class-based calibration. The corresponding standards, calibration curves, and quantification mode for each compound are provided in Supplementary Data 18.

PCA and OPLS-DA were performed for multivariate analysis to explore metabolite variation patterns between groups (biological replicates ≥3). VIP values from OPLS-DA combined with univariate P-value/FDR and FoldChange were used to identify significantly differential metabolites. Hierarchical cluster analysis (HCA) results of both samples and metabolites are presented as heatmaps with dendrograms. HCA was carried out with the R package Pheatmap, where normalized signal intensities of metabolites (Z-score normalization) are visualized as a color spectrum. Significantly differentially expressed metabolites between groups were determined based on criteria of |log2FoldChange| ≥ 1.0 and P ≤ 0.05. The ten most abundant metabolites in the black pseudo-stem (Mchb) of M. cheesmanii were selected for comparison with those in green pseudo-stems. Two-sided Student’s t-test analysis was further included for six target metabolites.

Statistics and reproducibility
Field experiment data were analyzed using one-way ANOVA followed by Duncan’s multiple range test, with differences considered significant at P < 0.05. For omics analyses (transcriptome and metabolomics) and qPCR experiments, three biological replicates were included. Statistical significance was assessed using a two-sided Student’s t-test at P < 0.05 (*), P < 0.01 (**), P < 0.001 (***). For comparative analysis of metabolite composition between Musa species, two phylogenetically closely related species were selected. No statistical method was employed to predetermine sample size, and all data were included in the analyses without exclusion.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors provide an interesting field and genetic study of the possibility of using M. cheesmanii as a potential wild crop relative to create new banana hybrids that may be resistant to different strains of Fusarium wilt (especially the virulent TR4). The paper also provides a range of background information on the genetics of banana evolution, genetic variation in eBSV and RGA2, and a multi-omics insight into black pseudostem coloration. These three sections are interesting and provide supporting information relevant to the use of M. cheesmanii as a potential WCR source for new hybrids.\nI especially commend the authors' intent to use more traditional crop breeding techniques involving WCRs as a means of generating new hybrids/crosses, rather than gene editing.\nThere are some methodological issues with the paper as written:\n1. The first results section provides insights into the susceptibility of crop progenitor species to most modern cultivars - essentially AA, AB, BB, AAB, ABB and AAA - with triploids dominating modern subsistence and cash crop cultivation globally. However, and somewhat oddly given the subsequent direction of the paper, a Cavendish cultivar (AAA) is chosen to demonstrate the susceptibility of cultivars to Foc_TR4. Given that M. cheesmanii is B clade/genome, and given the rest of the paper deals with two new hybrids derived from ABB cultivars, it would make sense to present data here for those hybrids susceptibility to Foc_TR4. Incidentally, it may also be worth discussing the pre-existing contribution of M. cheesmanii to some AAB and ABB hybrids - as I believe there is some.\n2. Throughout the paper, M.a ssp. malaccensis is used as the main Musa acuminata exemplar, which is okay - but would Musa acuminata ssp. banksii be more pertinent - as potentially it is the original M. acuminata domesticate that contributes especially to AAA cultivars and others (following a host of CIRAD genetics papers). It would seem that the role of M. a. ssp. mal. is later in the derivation of major cultivar groups - although some of the northerly AA and AAA cultivars, as well as some AAB and ABB have contributions from the westerly M. a ssp. Even though there may be some debate about all this, regarding the relative contributions of different M. a. ssp, the choice of M.a. ssp. mal as the representative of the A genome contribution to modern cultivars should be more clearly explained.\n3. lines 193-197 - a range of value judgements are made here regarding the growing and fruiting quality of the new derived hybrids, which are problematic. Why state 'best offspring' - rather just delete the word 'best' and retain the enhanced traits aspect (which you then state). Regarding fruit - and this recurs throughout the paper - you say 'excellent taste' (line 197) without providing any evidence for this. As raised in the paper, one of the major problems with banana breeding programs has been the derivation of new cultivars that are Foc resistant, while also retaining fruit quality. the latter is the hardest aspect to breed for - such that it is commercially viable (ie, as a replacement for the Foc susceptible Cavendish). I think we need more here to establish why the fruit quality is excellent - how measured - what was the control group and so on. Alternatively, maybe leave this as something to be determined through fruit quality tests - but state that the initial results are promising.\n4. lines 271-278 - the link of banana evolution to glacial cycles over the last 800,000 years or so is extremely tenuous. Your figure 3c indicates three glacial events for the last <800,000 years, whereas there have been 8-9 glacial cycles in that time - so why these three? Furthermore, the actual logic of the argument is limited - as the effects of glacial cycles are usually felt at higher altitudes and latitudes, rather than in lowland tropical locales (where effects can be somewhat more muted). It may affect mid-altitude species and also those on exposed continental shelfs during lower sea levels, but that is quite an argument to make (and it is not made). So, currently, the invocation of glacial cycles to understand banana demographic history/genetics is selective (why 3 not 7-8 glacial cycles) and requires justification (what is the process?).\n5. Generally the figures are of a high quality and informative. My only concerns focus on Figure 3 - which is hard to follow, even with the caption.\n- I think the figure needs to be rearranged - as effectively panel d could go at the end of the two panels in b (and why two panels in b) - as the three panels in total (two in b and one in d) actually correspond and flow from one another.\n- the different ordering of chromosomes in panel a and d is confusing - and I think the chromosomes in panel d should be labelled to avoid any confusion (as these just increase from 1,2, 3 and so on, whereas those in panel a do not).\n- again, panel c - I am not sure that the glacial cyclicity element works here - and if the glacial cycles are taken off - what are you trying to show? Maybe state it more clearly. Also, your caption implies the diagram shows the reduction in effective population size during the last 400-500,000 years in A genome species most markedly. However, why associated with glacial cyclicity - as effective population sizes for most species increase markedly before this reduction - and yet this is still within the period of Pleistocene glacial cyclicity (over 20 within the last 2.6 million years).\nThese are my substantive comments. I am not a geneticist and so cannot comment on the genetics methods and detail, but I hope my comments are useful for refining the logic of argument to the general readership of this journal.", "reviewer_2": "I read the manuscript titled \"Going wild in banana breeding: the cross-compatibility and multi-omics of Musa cheesmanii\" with great interest. I should underline that my expertise is mainly in genome assembly rather than banana genomics. I would like to congratulate the authors on this genome assembly. Even though it is of low complexity, they generated a substantial amount of diverse sequencing data and produced a high-quality reference genome for Musa cheesmanii. Here are my main concerns about this study.\nSequencing Data and Assembly Methods:\nThe authors generated interesting sequencing data and combined two long-read technologies with Hi-C data, which is better than the current standard. However, they used methods that are generally not employed for reference genomes: NextPolish and TGS-GapCloser. Most genomes from projects like VGP, dToL, ATLASea, or BGE are based on purge_dup (instead of Purge_Haplotigs), YaHS (instead of 3D-DNA), and are manually curated using Pretext (instead of Juicebox). This is not a problem at all, but the authors should provide adequate validation of their assembly at different stages.\nI would expect a supplementary table containing standard analyses such as BUSCO, Merqury, and KAT for the assembly obtained at each step: raw assembly, polished assembly, purged assembly, gap-closed assembly, and final assembly. This would allow monitoring the impact of each tool on the assembly.\nUse of TGS-GapCloser:\nThe authors used TGS-GapCloser, which is rarely used in the generation of reference genomes. I understand the temptation to get an assembly without gaps, but I would be very cautious about the accuracy of these gap-closed regions. The authors should clearly indicate their position in the genome and provide at least local validation using long-read coverage and Hi-C map visualization.\nChromosomal Inversion:\nI was curious to know if the inversion detected in chromosome 5 with Macuminata and Mschizocarpa occurs at the same position in Musa cheesmanii. Can the authors provide more information and details about this inversion? Validation of breakpoints would be of interest.\nData Availability:\nThe assembly and gene annotation were not available from the NGDC database nor from the Banana Genome Hub. Please provide access to the genome.\nMinor Points:\nLine 143: Is 34.3 days correct? Should it be less?\nLines 212-214: The analysis of GC content along the chromosomes is not a standard method to assess the quality of an assembly. Please remove this sentence.\nLines 233-240: This paragraph is a bit redundant and can be reduced.\nSection on Structural Variations (Lines 279-303): This section is very descriptive and can be reduced without changing the scientific content of the article.\nLines 610-613: Please add more description on the Hi-C library preparation.\nLine 640: The telomeric sequence in plants is T3AG3, not T3G3A as mentioned.", "reviewer_3": "Review of Manuscript ID NCOMMS-25-27107-T by Liu and colleagues.\nManuscript title: Going wild in banana breeding: the cross-compatibility and multi-omics of Musa cheesmanii\nThe manuscript presents a detailed investigation into the crossbreeding potential of Musa cheesmanii, highlighting its resistance to Fusarium wilt TR4 and its value as a male parent in banana improvement. The study also contributes a high-quality telomere-to-telomere genome assembly and complementary multi-omics data, providing useful insights into genome evolution and pigmentation traits in Musa species. The findings will be of interest to those working in banana breeding and genomics. However, the manuscript, in its current form, does not align with the broad conceptual scope and interdisciplinary impact required by Nature Communications. We recommend considering resubmission to a more specialised journal focused on crop improvement, plant genomics, or biotechnology, where the work will be more appropriately positioned.\nPlease see our comments below for further improvement.\nThank you.\nGeneral comments\nThis study contributes meaningfully to banana breeding research by integrating crossbreeding experiments, high-quality genomic data, and multi-omics analyses. The successful hybridisation of Musa cheesmanii with ABB cultivars highlights its potential to enhance disease resistance and yield. Notably, the authors report the first telomere-to-telomere (T2T) genome assembly for this species, which adds a valuable resource to the Musa genomics community.\nThe comparative genomic analysis provides useful insights into chromosomal rearrangements and structural variation among Musa species. Additionally, the integration of transcriptomic and metabolite profiling to investigate pseudostem pigmentation adds depth to the study. The anthocyanin pathway analysis offers a novel angle, although its biological significance remains unclear and may require further refinement.\nThe rationale for investigating pseudostem colouration is not convincingly presented, and the relevance of this trait to banana breeding is not well established. A more compelling case could have been made by prioritising traits directly linked to agronomic performance, such as disease resistance. For example, a comparative evolutionary analysis of resistance (R) gene families between M. cheesmanii and other Musa species with available high-quality genomes would have added greater value and relevance.\nOverall, the manuscript is well-structured, with a logical flow from the problem statement to actionable conclusions, supported by data across multiple levels of analysis.\nThe use of the first person “we” should be avoided.\nAbstract\nThe abstract provides a clear overview of the study’s objectives and highlights key outcomes, including Fusarium wilt resistance and the generation of productive hybrids. However, the manuscript lacks quantitative data to support several claims in the abstract, particularly those related to hybrid traits such as extended shelf life. Including specific metrics would enhance the credibility of these statements. Furthermore, phrases such as “breakthrough in genetic diversity” are overstated and not fully justified by the results presented. The expression “fully completed genome” is redundant; more precise terminology such as “gap-free” or “telomere-to-telomere (T2T) genome assembly” is recommended.\nIntroduction\nThe introduction provides a relevant overview of banana breeding challenges and the limited genetic diversity in modern cultivars. The inclusion of Fusarium wilt is appropriate, although it should be explicitly stated that Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) is currently the most devastating fungal disease affecting global banana production. The challenges associated with the activation of endogenous Banana streak virus (eBSV) sequences in hybrids are briefly mentioned, but their impact is understated. In practice, the risk of eBSV activation severely limits the use of B-genome cultivars in breeding programs, and this critical constraint deserves greater emphasis.\nThese issues help frame the rationale for exploring wild relatives like Musa cheesmanii as alternative genetic resources. However, the specific knowledge gap addressed by this study is not clearly defined. It remains unclear whether M. cheesmanii has never been previously used in breeding programs, or whether this work represents the first comprehensive analysis of its cross-compatibility and genomic potential.\nThe claim that this study presents “the first fully completed genome in banana” is unsubstantiated and should be revised. Similar high-contiguity, telomere-to-telomere assemblies have already been published for other Musa species, and this work should be positioned in relation to that context.\nFinally, the rationale for focusing exclusively on anthocyanins as the drivers of stem pigmentation is weak. Other phenolic compounds, particularly those that contribute to dark or black coloration through oxidative polymerisation (e.g. catechols, L-DOPA, caffeic acid), should also be considered. A more balanced introduction would acknowledge these alternative pathways and set the stage for a broader investigation of pigmentation mechanisms.\nResults\nThis section presents a substantial amount of data, but its impact is limited by gaps in quantitative reporting, inconsistent methodology, and several unsupported claims. While genome assembly and comparative genomics are handled well, data presentation regarding hybrid performance, pigmentation, and qPCR validation require some revision.\nPhenotypic evaluation and hybrid performance\n• Table 2 lacks critical information. It does not include plant height data, sample sizes, replication numbers, or any measures of variability such as standard deviations. Additionally, the parental line data are absent, making it difficult to contextualise and interpret the performance of the hybrids.\n• The number of plants evaluated for each hybrid and parental line should be explicitly reported in both the table and the main text.\n• Terms such as “normal seedling percentage” and “seed generation percentage” (lines 173-175) are vague and require clearer definitions to ensure the data are interpretable to readers unfamiliar with the breeding system described.\n• The shelf-life claim on line 197, which states an “extended shelf life of 6-9 days,” is not supported by quantitative data. This claim should be backed with comparative measurements between hybrids and parent lines. Similarly, the reference to “excellent taste” is subjective and should be removed unless supported by a sensory evaluation using an established methodology.\nDisease resistance data\n• While Table 1 presents disease resistance data clearly for the parental lines, no equivalent data are provided for the hybrids. This omission limits the strength of the claim that resistance traits have been successfully transferred.\n• A new table should be added to report disease resistance data for the hybrids using the same scoring or index system used in Table 1.\n• The “Disease Index” formula referenced in Table 1 should be added to the “Methods” section at least, to ensure transparency and reproducibility.\n• The use of “NS” to denote “no symptoms” is inconsistent across the table as NS also appear in the IP column.\nMetabolite analysis and pigmentation\n• The authors report the detection of numerous low-abundance anthocyanins (Figure S18). However, the authors have not provided any detail in their methods to evaluate the reliability of their compound identification or quantification. Critical information missing includes sample preparation protocols, chromatographic separation parameters, MS/MS acquisition settings, and criteria used for compound identification. This lack of transparency is particularly concerning given that several anthocyanins share identical m/z values, making chromatographic resolution and MS/MS spectral interpretation essential for accurate compound differentiation. For metabolite quantification the authors should report whether they used any standards for quantification. Without this information, it is difficult to assess whether the low-abundance signals are from true analytes or are analytical artifacts.\n• Following this, the authors suggest that the black stem colouration is due to accumulation of specific anthocyanins presented in Supplementary figure 18b. Whilst the authors have reported statistical significance in compound accumulation, given their extremely low abundance (for example 0.02 ug/g pelargonidin-3-O-arabinoside) compared to other identified compounds (eg. in S18a procyanidin B2 is reported at around 300 ug/g for both M. cheesmanii and the green stemmed Musa acuminata) is this difference likely to be biologically relevant in creating the black stem phenotype? The authors should note that dark brown or black colouration can come from sources other than anthocyanins. Oxidation of phenolics which have two neighbouring hydroxyl groups can lead to the formation of quinone-like structure which can further undergo polymerisation making brown/black colouration. Examples include caffeic acid, catechol, catechin, dopamine and L-DOPA. Dopamine and L-DOPA have previously been detected in various banana tissues at high concentrations. Additionally, as the first 3 compounds share many of the genes/enzymes with the anthocyanin biosynthesis pathway, the authors should consider adjusting the manuscript (including transcriptome analysis) to encompass all phenolics rather than only anthocyanins.\nTranscriptomic validation (qPCR)\n• The claim that transcriptomic findings were validated by qPCR (lines 377-378) is misleading. For example, the gene F3’H is not statistically significant based on Supplementary Figure 15a, and there is no presented qPCR data for PAL2 or BGLU26, even though these genes are mentioned.\n• The qPCR methodology is also not described. The manuscript should include details about cDNA synthesis protocols, primer sequences, and amplification conditions in the “Methods” section.\n• To support transparency and reproducibility, the authors should also consider providing raw Ct values as a supplementary table.\n• Supplementary Figure 8a appears to be nearly identical to Figure 4a in the main text. To avoid redundancy, one of these figures should be removed.\nMinor revisions and editorial suggestions\n• The authors should ensure consistent terminology and formatting across all tables and figures, particularly for labels and abbreviations.\n• All abbreviations should be defined upon first use in each table or figure, even if they are explained elsewhere in the manuscript. For example, “eBSV” should be redefined as “endogenous banana streak virus” where it first appears.\nDiscussion\nThis is a good discussion supported by a solid foundation of relevant literature.\nCertain sections, such as the paragraph beginning at line 509, primarily summarise results rather than examining their broader implications within the context of existing research.\nThe discussion does not consider some of the practical challenges associated with the use of M. cheesmanii in breeding programs. These may include low hybrid recovery rates, the potential for linkage drag, and the complexity of backcrossing strategies. Additionally, phrases such as “revolutionary breakthrough” and “remarkable productivity” are used in the text without sufficient supporting evidence and do not align with the otherwise analytical tone.\nExpanding the discussion to include resistance genes, specifically identifying the types of loci involved, could add value. Comparing these loci to those found in other published Musa genomes, and considering their potential implications for breeding strategies, would provide additional depth and relevance.\nThe structure of the discussion may be improved by the inclusion of subheadings to support clarity and assist reader navigation. Several paragraphs are lengthy and would benefit from being divided into smaller units. Although the intended audience is likely to have technical expertise, certain terms could be clarified to increase accessibility. For example, “eBSV” could be restated as “endogenous banana streak virus,” and terms such as “segregation distortion” and “structural heterozygosity” could be accompanied by short explanations.\nThe presentation of data may be strengthened through brief agronomic interpretation. For instance, a girth to height ratio of 0.225 could be described as exceeding the threshold associated with resistance to lodging.\nFinally, the discussion could also benefit from consideration of future directions, such as whether similar traits may be found in other underutilised Musa species or what risks could emerge from the widespread deployment of M. cheesmanii in breeding programs.\nMethods\nCritical methodological details are missing in several areas, which limits the reproducibility and rigour of the study.\nThe description of Fusarium oxysporum f. sp. cubense TR4 inoculation lacks essential information. The origin of the fungal strain, its vegetative compatibility group (VCG), and the inoculation protocol (particularly the spore concentration and method of application) are not provided. In addition, the criteria used to evaluate external and internal symptoms of infection, and how disease severity was scored or calculated, are not adequately described.\nHybrid evaluation is similarly underdeveloped. Terms such as “seed set,” “seed generation,” and “normal seedling” (lines 592-593) are ambiguous and require precise definitions. The section describing hybrid assessment (lines 592-597) lists variables that were evaluated but does not explain how these measurements were conducted. Sample sizes for hybrid offspring are also not reported, and replication details are entirely missing.\nThe anthocyanin analysis lacks transparency, with key methodological details missing. Specific recommendations for improving this section are outlined in the Results critique above.\nqPCR validation of transcriptomic data is not described at all. There is no information about cDNA synthesis, primer sequences, qPCR reaction conditions, or normalisation strategies. To improve transparency and reproducibility, the authors should include these details in the methods and provide raw Ct values in a supplementary table.\nFinally, a specific section on statistical analysis should be included.\nTables and figures\n• The overall quality and readability of the tables and figures is good. However, Figures 1 and 2 would benefit significantly from higher-resolution images and improved formatting to enhance visual clarity.\n• In Figure 2, scale bars are missing from all panels and should be added to ensure interpretability, particularly for morphological comparisons.\n• In Table 2, the definition of “plant stature” is placed in the footnotes, should also be added to the main text (Methods). Table 2 needs to include data from parental lines, plant height measurements, standard deviations, and the number of replicates. A new table reporting hybrid disease resistance should be created, modelled on Table 1.\n• Figure 4, panel (e), which presents RGA2 polymorphisms, requires clearer labelling to highlight the nonsense mutation (T→A at 3,687 bp).\n• Figure 4 and Supplementary Figure 8 present nearly identical phylogenetic trees. To avoid redundancy, one of these should be removed.\n• Figure legends throughout should be made more descriptive, especially when referring to multi-panel figures.\nEnd of report", "reviewer_4": "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."}, "author_response": "Response to reviewers\nReviewer #1:\nThe authors provide an interesting field and genetic study of the possibility of using\nM. cheesmanii as a potential wild crop relative to create new banana hybrids that may\nbe resistant to different strains of Fusarium wilt (especially the virulent TR4). The\npaper also provides a range of background information on the genetics of banana\nevolution, genetic variation in eBSV and RGA2, and a multi-omics insight into black\npseudostem coloration. These three sections are interesting and provide supporting\ninformation relevant to the use of M. cheesmanii as a potential WCR source for new\nhybrids.\nI especially commend the authors' intent to use more traditional crop breeding\ntechniques involving WCRs as a means of generating new hybrids/crosses, rather than\ngene editing.\nThank you for your positive feedback and for recognizing the relevance of our work.\nWe also sincerely appreciate your constructive suggestions. We have made every\neffort to revise the manuscript accordingly and hope the updated version addresses\nyour comments satisfactorily.\nThere are some methodological issues with the paper as written:\n1. The first results section provides insights into the susceptibility of crop progenitor\nspecies to most modern cultivars - essentially AA, AB, BB, AAB, ABB and AAA -\nwith triploids dominating modern subsistence and cash crop cultivation globally.\nHowever, and somewhat oddly given the subsequent direction of the paper, a\nCavendish cultivar (AAA) is chosen to demonstrate the susceptibility of cultivars to\nFoc_TR4. Given that M. cheesmanii is B clade/genome, and given the rest of the\npaper deals with two new hybrids derived from ABB cultivars, it would make sense to\npresent data here for those hybrids susceptibility to Foc_TR4. Incidentally, it may\nalso be worth discussing the pre-existing contribution of M. cheesmanii to some AAB\nand ABB hybrids - as I believe there is some.\nThank you for this constructive suggestion. We have supplemented the manuscript\nwith resistance evaluation data for both the hybrids and their parental lines. The\nresults show that the hybrid offspring exhibited enhanced resistance compared to their\nrespective maternal parents (Table 1, Fig. 1). Notably, M. cheesmanii and its hybrid\noffspring 'Haijiao No. 1' (M. cheesmanii × 'Yulin') were classified as highly resistant\n(lines 168-181). In addition, we selected the Silk and Plantain cultivars to assess the\npotential genetic contribution of M. cheesmanii to these hybrids, as these two AAB\ncultivars are currently the only ones with high-quality reference genomes that contain\nB-genome components. We combined potential wild species reference genomes as\nancestral references and split the Silk and Plantain genomes into 2-kb fragments,\nwhich were then re-mapped to the ancestral reference genomes. The most similar\nmatches were used to infer their likely origins. As shown in Supplementary Fig. 5, M.\ncheesmanii contributes very little, if at all, to these two common hybrids. This\nsuggests that M. cheesmanii has not yet been used in breeding programs for these\ncultivars, highlighting its undervalued potential for future breeding. Corresponding\ndescriptions have been added to the main text (lines 251-254), and detailed methods\nare provided in the Methods section (lines 765-769).\n2. Throughout the paper, M.a ssp. malaccensis is used as the main Musa acuminata\nexemplar, which is okay - but would Musa acuminata ssp. banksii be more pertinent -\nas potentially it is the original M. acuminata domesticate that contributes especially to\nAAA cultivars and others (following a host of CIRAD genetics papers). It would\nseem that the role of M. a. ssp. mal. is later in the derivation of major cultivar groups -\nalthough some of the northerly AA and AAA cultivars, as well as some AAB and\nABB have contributions from the westerly M. a ssp. Even though there may be some\ndebate about all this, regarding the relative contributions of different M. a. ssp, the\nchoice of M.a. ssp. mal as the representative of the A genome contribution to modern\ncultivars should be more clearly explained.\nThis is an important question. Considering the Cavendish 'Baxijiao', Musa acuminata\nssp. malaccensis is one of several M. acuminata subspecies contributing to its\nA-genome, accounting for approximately 21% of the genome. In contrast, banksii\ncontributes the most (~43%), followed by zebrina (~25%), with the remainder coming\nfrom other subspecies (Martin et al., 2020, 2023; Sardos et al., 2025). With regards to\nABB Pisang Awak, malaccensis is the main contributor from the A genome. In\ngeneral, since banksii already contributes heavily to the genomes of most AAA, AAB,\nand ABB bananas, its diversity is already well represented and lack some interesting\ntraits, so it has not been prioritized for breeding. In contrast, malaccensis is often the\nmost cited subspecies because it was used for the first reference A genome, and has\nbeen studied more extensively, particularly for its resistance to Fusarium oxysporum f.\nsp. cubense (Foc) tropical race 4 (Foc_TR4). The RGA2 gene, linked to Foc_TR4\nresistance, was identified in fact in malaccensis (Dale et al., 2017). Since our\nmanuscript discusses both Foc_TR4 considerations and RGA2, we highlighted\nmalaccensis more prominently. However, based on your comment, we have now\nclarified the relative contributions of the different subspecies in the manuscript.\nLine 67 we added: Most of the observed variation arises from variable mosaic\ncontributions of different Musa acuminata subspecies.\nLine 89 we added: Musa acuminata comprises several subspecies, including banksii,\nmalaccensis, zebrina, and burmannica. Among them, M. acuminata ssp. banksii is the\nmajor contributor to all cultivated bananas followed M. acuminata ssp. zebrina.\nAlthough M. acuminata ssp. burmannica, particularly the accession 'Calcutta 4', is not\ncommonly found in natural cultivars, it has been widely used in breeding programs\ndue to its strong disease resistance. Notably, malaccensis is also known for its high\nresistance to Foc, as well as a significant contributor to several cultivar groups,\nincluding AAA (e.g., Cavendish, Gros Michel, Red), AAB (e.g., Pome, Silk), and\nABB (e.g., Pisang Awak).\nMartin, G., Cardi, C., Sarah, G., Ricci, S., Jenny, C., Fondi, E., ... & Yahiaoui, N.\n(2020). Genome ancestry mosaics reveal multiple and cryptic contributors to\ncultivated banana. The Plant Journal, 102(5), 1008-1025.\nMartin, G., Cottin, A., Baurens, F. C., Labadie, K., Hervouet, C., Salmon, F., ... &\nYahiaoui, N. (2023). Interspecific introgression patterns reveal the origins of\nworldwide cultivated bananas in New Guinea. The Plant Journal, 113(4), 802-818.\nSardos, J., Cenci, A., Martin, G., Breton, C., Guignon, V., Van den Houwe, I., ... &\nRouard, M. (2025). Painting the diversity of a world's favorite fruit: A next generation\ncatalog of cultivated bananas. Plants, People, Planet, 7(1), 263-283.\nDale, J., James, A., Paul, J. Y., Khanna, H., Smith, M., Peraza-Echeverria, S., ... &\nHarding, R. (2017). Transgenic Cavendish bananas with resistance to Fusarium wilt\ntropical race 4. Nature communications, 8(1), 1496.\n3. lines 193-197 - a range of value judgements are made here regarding the growing\nand fruiting quality of the new derived hybrids, which are problematic. Why state\n'best offspring' - rather just delete the word 'best' and retain the enhanced traits aspect\n(which you then state). Regarding fruit - and this recurs throughout the paper - you\nsay 'excellent taste' (line 197) without providing any evidence for this. As raised in\nthe paper, one of the major problems with banana breeding programs has been the\nderivation of new cultivars that are Foc resistant, while also retaining fruit quality. the\nlatter is the hardest aspect to breed for - such that it is commercially viable (ie, as a\nreplacement for the Foc susceptible Cavendish). I think we need more here to\nestablish why the fruit quality is excellent - how measured - what was the control\ngroup and so on. Alternatively, maybe leave this as something to be determined\nthrough fruit quality tests - but state that the initial results are promising.\nThank you for this helpful comment. The word “best” has been removed as suggested;\nthe sentence now reads: “Significant heterobeltiosis was observed in the M.\ncheesmanii × 'Yulin' cross, with the hybrid showing complementary traits.”\nRegarding fruit quality, we have added evaluation results, including sensory\nassessment, chemical composition analysis, and shelf-life determination. The results\nshow that the hybrid offspring 'Haijiao No. 1' (M. cheesmanii × 'Yulin') exhibited\nimproved quality compared with its maternal parent 'Yulin' (see Supplementary\nTables 1-2 and Supplementary Fig. 1). In addition, we have revised the text to ensure\na more objective description, for example, “superior performance” has been replaced\nwith “enhanced performance”, and “excellent taste” has been revised to “favorable\ntaste”.\n4. lines 271-278 - the link of banana evolution to glacial cycles over the last 800,000\nyears or so is extremely tenuous. Your figure 3c indicates three glacial events for the\nlast <800,000 years, whereas there have been 8-9 glacial cycles in that time - so why\nthese three? Furthermore, the actual logic of the argument is limited - as the effects of\nglacial cycles are usually felt at higher altitudes and latitudes, rather than in lowland\ntropical locales (where effects can be somewhat more muted). It may affect\nmid-altitude species and also those on exposed continental shelfs during lower sea\nlevels, but that is quite an argument to make (and it is not made). So, currently, the\ninvocation of glacial cycles to understand banana demographic history/genetics is\nselective (why 3 not 7-8 glacial cycles) and requires justification (what is the\nprocess?).\nWe agree that the evidence linking banana evolution directly to glacial cycles in our\noriginal submission was insufficient and somewhat selective. In the revised\nmanuscript, we have clarified that over the past ~1 million years there have been\napproximately nine glacial-interglacial cycles, rather than only the three highlighted\nin our initial Figure 3c. We now focus on the contrasting demographic histories of\ntwo major clades: Clade A (M. acuminata and M. schizocarpa) and Clade B (M.\nbalbisiana and M. cheesmanii). We emphasize that Clade A shows more pronounced\nfluctuations in effective population size (Ne) corresponding to glacial-interglacial\ncycles, whereas Clade B exhibits relatively stable Ne, suggesting greater resilience to\nenvironmental variability. Please see lines 305-318 and the revised Figure 3c.\n5. Generally the figures are of a high quality and informative. My only concerns focus\non Figure 3 - which is hard to follow, even with the caption.\n- I think the figure needs to be rearranged - as effectively panel d could go at the end\nof the two panels in b (and why two panels in b) - as the three panels in total (two in b\nand one in d) actually correspond and flow from one another.\n- the different ordering of chromosomes in panel a and d is confusing - and I think the\nchromosomes in panel d should be labelled to avoid any confusion (as these just\nincrease from 1,2, 3 and so on, whereas those in panel a do not).\n- again, panel c - I am not sure that the glacial cyclicity element works here - and if\nthe glacial cycles are taken off - what are you trying to show? Maybe state it more\nclearly. Also, your caption implies the diagram shows the reduction in effective\npopulation size during the last 400-500,000 years in A genome species most markedly.\nHowever, why associated with glacial cyclicity - as effective population sizes for\nmost species increase markedly before this reduction - and yet this is still within the\nperiod of Pleistocene glacial cyclicity (over 20 within the last 2.6 million years).\nWe appreciate your constructive comments on Figure 3. The figure has been revised\naccordingly based on the content adjustments in the manuscript.\nThese are my substantive comments. I am not a geneticist and so cannot comment on\nthe genetics methods and detail, but I hope my comments are useful for refining the\nlogic of argument to the general readership of this journal.\nThank you for your constructive comments, which are very helpful for refining the\nclarity and logic of our manuscript for a broader readership.\nReviewer #2:\nI read the manuscript titled \"Going wild in banana breeding: the cross-compatibility\nand multi-omics of Musa cheesmanii\" with great interest. I should underline that my\nexpertise is mainly in genome assembly rather than banana genomics. I would like to\ncongratulate the authors on this genome assembly. Even though it is of low\ncomplexity, they generated a substantial amount of diverse sequencing data and\nproduced a high-quality reference genome for Musa cheesmanii. Here are my main\nconcerns about this study.\nWe sincerely thank you for your thoughtful evaluation of our manuscript and are\nencouraged that you consider our reference genome to be of high quality despite the\nrelatively low complexity of this species. We also thank you for raising important\nconcerns, which we have carefully considered and addressed in the revised\nmanuscript as detailed in our point-by-point responses below.\nSequencing Data and Assembly Methods:\nThe authors generated interesting sequencing data and combined two long-read\ntechnologies with Hi-C data, which is better than the current standard. However, they\nused methods that are generally not employed for reference genomes: NextPolish and\nTGS-GapCloser. Most genomes from projects like VGP, dToL, ATLASea, or BGE\nare based on purge_dup (instead of Purge_Haplotigs), YaHS (instead of 3D-DNA),\nand are manually curated using Pretext (instead of Juicebox). This is not a problem at\nall, but the authors should provide adequate validation of their assembly at different\nstages.\nI would expect a supplementary table containing standard analyses such as BUSCO,\nMerqury, and KAT for the assembly obtained at each step: raw assembly, polished\nassembly, purged assembly, gap-closed assembly, and final assembly. This would\nallow monitoring the impact of each tool on the assembly.\nThank you for this valuable suggestion. As recommended, we have performed\nassembly evaluations at different stages of the pipeline, including the primary HiFi\nassembly, polished primary assembly, purged assembly, Hi-C anchored assembly,\ngap-closed assembly, and the final assembly. For each stage, we assessed the quality\nusing BUSCO, Merqury, and KAT in order to monitor the impact of each tool on\nassembly quality. The results show consistently high performance across all stages.\nDetailed results are now provided in Supplementary Table 6 and Supplementary Fig.\n3.\nUse of TGS-GapCloser:\nThe authors used TGS-GapCloser, which is rarely used in the generation of reference\ngenomes. I understand the temptation to get an assembly without gaps, but I would be\nvery cautious about the accuracy of these gap-closed regions. The authors should\nclearly indicate their position in the genome and provide at least local validation using\nlong-read coverage and Hi-C map visualization.\nThank you for this important comment. We agree that careful validation of gap-closed\nregions is essential. In response, we have clearly marked the positions of all four\npreviously gapped regions in the figures used for visual validation. We examined the\nbreakpoint regions using long-read coverage and confirmed that the sequences used to\nclose the gaps span the breakpoints. We also added Hi-C heatmaps to support the\nassembly. However, we would like to note that gap4 is located in a highly repetitive\nrDNA array region on chromosome 10. This type of complex region is commonly\nassociated with assembly breaks and is known to display strong internal Hi-C\ninteractions but weak connections to neighboring regions, making it difficult for Hi-C\ndata alone to resolve. Similar patterns have been observed in other Musa genome\nassemblies, including those of M. acuminata, M. balbisiana, and cultivated Cavendish\nbanana. Fortunately, our ONT long reads enabled us to confidently fill this region.\nRelevant supporting figures have been added (see Supplementary Figures 23-24).\nChromosomal Inversion:\nI was curious to know if the inversion detected in chromosome 5 with Macuminata\nand Mschizocarpa occurs at the same position in Musa cheesmanii. Can the authors\nprovide more information and details about this inversion? Validation of breakpoints\nwould be of interest.\nThe inversion on chromosome 5 observed in M. acuminata and M. schizocarpa is not\npresent in M. cheesmanii, as shown in Fig. 3b and Supplementary Fig. 9. In addition,\nwe have validated the detected structural variation breakpoints using long-read\nalignments (Supplementary Fig. 10), which confirm the presence of these structural\nvariations, including this inversion.\nData Availability:\nThe assembly and gene annotation were not available from the NGDC database nor\nfrom the Banana Genome Hub. Please provide access to the genome.\nThe assembly and gene annotation are available on Figshare:\nhttp://doi.org/10.6084/m9.figshare.25658835\nSpecifically, the genome assembly file is Mch.reverse.w0.renumbering.fasta, and the\ngenome annotation file is Mch.reverse.renumbering.gff.\nMinor Points:\nLine 143: Is 34.3 days correct? Should it be less?\nWe have confirmed that 34.3 days is correct. In our previous study evaluating\nFusarium wilt resistance in 'Baxijiao' (Li et al., 2015), the incubation period was\nreported as 33.1 days. The close agreement between the two results supports the\nreliability of the incubation period value in this study.\nLi, W. M., Dita, M., Wu, W., Hu, G. B., Xie, J. H., & Ge, X. J. (2015). Resistance\nsources to Fusarium oxysporum f. sp. cubense tropical race 4 in banana wild relatives.\nPlant Pathology, 64(5), 1061-1067.\nLines 212-214: The analysis of GC content along the chromosomes is not a standard\nmethod to assess the quality of an assembly. Please remove this sentence.\nThis sentence has been removed.\nLines 233-240: This paragraph is a bit redundant and can be reduced.\nThank you for the suggestion. We have revised this paragraph to remove redundancy\nand improve clarity, while retaining all key data and references (Lines 271-278).\nSection on Structural Variations (Lines 279-303): This section is very descriptive and\ncan be reduced without changing the scientific content of the article.\nThank you for the suggestion. We have revised this section to reduce descriptive\ndetail and improve conciseness, while retaining the key scientific content (Lines\n319-338).\nLines 610-613: Please add more description on the Hi-C library preparation.\nThank you for the suggestion. A brief description of the Hi-C library preparation steps\nhas been added (Lines 732-734).\nLine 640: The telomeric sequence in plants is T3AG3, not T3G3A as mentioned.\nThank you for pointing out the error regarding the telomeric sequence. We have\ncorrected it from T3G3A to T3AG3 in the manuscript accordingly.\nReviewer #3:\nReview of Manuscript ID NCOMMS-25-27107-T by Liu and colleagues.\nManuscript title: Going wild in banana breeding: the cross-compatibility and\nmulti-omics of Musa cheesmanii\nThe manuscript presents a detailed investigation into the crossbreeding potential of\nMusa cheesmanii, highlighting its resistance to Fusarium wilt TR4 and its value as a\nmale parent in banana improvement. The study also contributes a high-quality\ntelomere-to-telomere genome assembly and complementary multi-omics data,\nproviding useful insights into genome evolution and pigmentation traits in Musa\nspecies. The findings will be of interest to those working in banana breeding and\ngenomics. However, the manuscript, in its current form, does not align with the broad\nconceptual scope and interdisciplinary impact required by Nature Communications.\nWe recommend considering resubmission to a more specialised journal focused on\ncrop improvement, plant genomics, or biotechnology, where the work will be more\nappropriately positioned.\nPlease see our comments below for further improvement.\nThank you.\nWe sincerely thank you for the thorough and constructive comments, which have\nbeen extremely valuable in improving our manuscript. Bananas are among the most\nimportant staple crops worldwide, yet they face the severe threat of Fusarium wilt.\nCultivated bananas already possess a narrow genetic base, and the predominant\nreliance on M. acuminata subspecies as breeding parents has further intensified this\nlimitation, leaving the crop even more vulnerable to diseases. Identifying new\nparental resources is therefore of critical importance. However, progress has been\nextremely limited for decades due to the crop's triploid nature (resulting in low\nfertility) and its lengthy breeding cycles. Through extensive resistance evaluations\nand crossbreeding efforts, we have identified M. cheesmanii as a promising candidate\nparent. Together with complementary multi-omics analyses, our work not only\naddresses urgent challenges in banana improvement but also provides essential\nbackground knowledge of this species. We have carefully revised the manuscript\naccording to your suggestions, and we hope that this version now meets the high\nstandards and interdisciplinary impact expected for publication in Nature\nCommunications.\nGeneral comments\nThis study contributes meaningfully to banana breeding research by integrating\ncrossbreeding experiments, high-quality genomic data, and multi-omics analyses. The\nsuccessful hybridisation of Musa cheesmanii with ABB cultivars highlights its\npotential to enhance disease resistance and yield. Notably, the authors report the first\ntelomere-to-telomere (T2T) genome assembly for this species, which adds a valuable\nresource to the Musa genomics community.\nThe comparative genomic analysis provides useful insights into chromosomal\nrearrangements and structural variation among Musa species. Additionally, the\nintegration of transcriptomic and metabolite profiling to investigate pseudostem\npigmentation adds depth to the study. The anthocyanin pathway analysis offers a\nnovel angle, although its biological significance remains unclear and may require\nfurther refinement.\nThe rationale for investigating pseudostem colouration is not convincingly presented,\nand the relevance of this trait to banana breeding is not well established. A more\ncompelling case could have been made by prioritising traits directly linked to\nagronomic performance, such as disease resistance. For example, a comparative\nevolutionary analysis of resistance (R) gene families between M. cheesmanii and\nother Musa species with available high-quality genomes would have added greater\nvalue and relevance.\nWe agree with this comment and complemented the study a comparative evolutionary\nanalysis of resistance (R) gene families between M. cheesmanii and other Musa\nspecies (see lines 339-361 and Figure 4).\nOverall, the manuscript is well-structured, with a logical flow from the problem\nstatement to actionable conclusions, supported by data across multiple levels of\nanalysis.\nThe use of the first person “we” should be avoided.\nWe have carefully reviewed the use of the first person “we” throughout the\nmanuscript and revised it where possible.\nAbstract\nThe abstract provides a clear overview of the study’s objectives and highlights key\noutcomes, including Fusarium wilt resistance and the generation of productive\nhybrids. However, the manuscript lacks quantitative data to support several claims in\nthe abstract, particularly those related to hybrid traits such as extended shelf life.\nIncluding specific metrics would enhance the credibility of these statements.\nFurthermore, phrases such as “breakthrough in genetic diversity” are overstated and\nnot fully justified by the results presented. The expression “fully completed genome”\nis redundant; more precise terminology such as “gap-free” or “telomere-to-telomere\n(T2T) genome assembly” is recommended.\nWe appreciate these helpful comments. We have incorporated quantitative data to\nsubstantiate the claims about the enhanced hybrid traits, revised overstated\nexpressions to ensure a more objective presentation, and replaced “fully completed\ngenome” with “telomere-to-telomere, gap-free genome assembly”.\nIntroduction\nThe introduction provides a relevant overview of banana breeding challenges and the\nlimited genetic diversity in modern cultivars. The inclusion of Fusarium wilt is\nappropriate, although it should be explicitly stated that Fusarium oxysporum f. sp.\ncubense tropical race 4 (TR4) is currently the most devastating fungal disease\naffecting global banana production. The challenges associated with the activation of\nendogenous Banana streak virus (eBSV) sequences in hybrids are briefly mentioned,\nbut their impact is understated. In practice, the risk of eBSV activation severely limits\nthe use of B-genome cultivars in breeding programs, and this critical constraint\ndeserves greater emphasis.\nThank you for your helpful comments. In response, we have made two key revisions\nin the Introduction to better highlight the issues you raised. First, to emphasize the\nthreat posed by Fusarium wilt, we added the sentence: “However, global production is\nincreasingly threatened by Fusarium wilt, a fungal disease caused by Fusarium\noxysporum f. sp. cubense (Foc), with tropical race 4 (Foc_TR4) currently recognized\nas the most devastating fungal pathogen affecting banana cultivation worldwide”\n(lines 54-57). Second, regarding the activation of eBSV, we revised the text to: “The\nuse of M. balbisiana is severely constrained by the risk of activating endogenous\nbanana streak virus (eBSV), especially under stress or during in vitro culture, which\ncan lead to spontaneous viral expression in hybrids and compromise plant health and\nyield. This genetic instability not only limits the use of B-genome cultivars in banana\nimprovement programs but also hampers global germplasm exchanges due to the risk\nof eBSV activation into an infectious episomal form” (lines 97-102). We believe these\nupdates better reflect the severity and practical implications of these challenges in\nbanana breeding.\nThese issues help frame the rationale for exploring wild relatives like Musa\ncheesmanii as alternative genetic resources. However, the specific knowledge gap\naddressed by this study is not clearly defined. It remains unclear whether M.\ncheesmanii has never been previously used in breeding programs, or whether this\nwork represents the first comprehensive analysis of its cross-compatibility and\ngenomic potential.\nThank you for highlighting this important point. As far as we are aware, there are no\npublished studies reporting the use of M. cheesmanii in banana breeding programs,\nnor comprehensive evaluations of its cross-compatibility or genomic potential. We\nalso selected the Silk and Plantain cultivars to assess the potential genetic contribution\nof M. cheesmanii to these hybrids, as these two AAB cultivars are currently the only\nones with high-quality reference genomes that contain B-genome components. As\nshown in Supplementary Fig. 5, M. cheesmanii contributes very little, if at all, to these\ntwo common hybrids. This suggests that M. cheesmanii has not yet been used in\nbreeding programs for these cultivars, highlighting its undervalued potential for future\nbreeding. To clarify the novelty and relevance of our work, we have revised the\nmanuscript accordingly to emphasize this knowledge gap: “However, M. cheesmanii\nhas not previously been utilized or systematically examined in banana breeding\nresearch, and its cross-compatibility and genetic characteristics remain unexplored,\nindicating the need for in-depth characterization to support its use in breeding” (lines\n125-129).\nThe claim that this study presents “the first fully completed genome in banana” is\nunsubstantiated and should be revised. Similar high-contiguity, telomere-to-telomere\nassemblies have already been published for other Musa species, and this work should\nbe positioned in relation to that context.\nThank you for pointing this out. We agree that our original wording was too strong.\nAlthough our Musa cheesmanii assembly is more complete than previously published\nMusa genomes (being gap-free and including all telomeric sequences), we have\nrevised the sentence in the manuscript to describe this more accurately. The updated\nsentence now reads: \"A telomere-to-telomere (T2T) gap-free genome assembly of M.\ncheesmanii is also presented, representing the most complete banana genome to date,\nwith all telomeres identified and no assembly gaps\" (lines 137-139).\nFinally, the rationale for focusing exclusively on anthocyanins as the drivers of stem\npigmentation is weak. Other phenolic compounds, particularly those that contribute to\ndark or black coloration through oxidative polymerisation (e.g. catechols, L-DOPA,\ncaffeic acid), should also be considered. A more balanced introduction would\nacknowledge these alternative pathways and set the stage for a broader investigation\nof pigmentation mechanisms.\nWe appreciate your valuable comment. We have revised the Introduction to\nacknowledge that, in addition to anthocyanins, the oxidative polymerization of\nphenolic compounds (e.g., catechols, L-DOPA, caffeic acid) may also contribute to\nthe dark pigmentation observed in banana pseudo-stems (lines 116-118).\nResults\nThis section presents a substantial amount of data, but its impact is limited by gaps in\nquantitative reporting, inconsistent methodology, and several unsupported claims.\nWhile genome assembly and comparative genomics are handled well, data\npresentation regarding hybrid performance, pigmentation, and qPCR validation\nrequire some revision.\nPhenotypic evaluation and hybrid performance\n• Table 2 lacks critical information. It does not include plant height data, sample sizes,\nreplication numbers, or any measures of variability such as standard deviations.\nAdditionally, the parental line data are absent, making it difficult to contextualise and\ninterpret the performance of the hybrids.\nWe appreciate your constuctive comment. Table 2 has been revised to include a\ncomplete set of data for both parental lines and hybrids. Plant height (here evaluate\nusing pseudostem height) and other key agronomic traits, including bunch weight, and\nplant stature, are now reported with mean values and standard deviations (mean ± SD).\nDetail information on sample sizes, replication numbers, and experimental design is\nprovided in both Table 2 and the Methods section (Please see lines 691-700).\n• The number of plants evaluated for each hybrid and parental line should be\nexplicitly reported in both the table and the main text.\nThe number of plants evaluated for each trait has now been reported in both the\nTables and the Methods section. For the resistance evaluation in Table 1, each\naccession was represented by 15 plants. Due to asynchronous development among\ndifferent accessions, the sample sizes are not identical across all traits in Table 2. For\nthe reproductive performance traits (seed set, seed germination and normal seedling),\nwe evaluated more than 3,000 fruits, 300 seeds, and 80 hybrid progenies, replicated\nthree times. Pseudostem colour was assessed in over 100 plants. For the agronomic\ntraits (bunch weight, pseudostem height, and plant stature), each parental line was\nrepresented by 10 randomly selected plants from population. For the F1 hybrid\ncombinations, considering differences in cross-compatibility as well as the stability\nand agronomic performance of F1 progeny phenotypes, we selected the 10 hybrid\nlines that exhibited the best fruit quality with each line represented by 5 plants.\nDetails are shown in Table 1 & 2, and lines 651-652, 696-700.\n• Terms such as “normal seedling percentage” and “seed generation percentage”\n(lines 173-175) are vague and require clearer definitions to ensure the data are\ninterpretable to readers unfamiliar with the breeding system described.\nThank you for this comment. The term “normal seedling” has now been clarified as\n“seedlings with healthy and regular appearance” (line 204). \"Seed set\" refers to the\naverage number of seeds produced per fruit (line 202). In addition, “seed generation\npercentage” has been corrected to “seed germination percentage” (line 202).\n• The shelf-life claim on line 197, which states an “extended shelf life of 6-9 days,” is\nnot supported by quantitative data. This claim should be backed with comparative\nmeasurements between hybrids and parent lines. Similarly, the reference to “excellent\ntaste” is subjective and should be removed unless supported by a sensory evaluation\nusing an established methodology.\nWe have added quantitative data on shelf-life determination, showing that the edible\nperiod of the hybrid 'Haijiao No. 1' (M. cheesmanii × 'Yulin') is longer than that of its\nmaternal parent 'Yulin' (Supplementary Fig. 1). In addition, a sensory evaluation was\nconducted, indicating that 'Haijiao No. 1' had a more favorable taste compared with\n'Yulin' (Supplementary Table 1). Nevertheless, to ensure objectivity, the term\n“excellent taste” has been replaced with “favorable taste” in the revised manuscript.\nDisease resistance data\n• While Table 1 presents disease resistance data clearly for the parental lines, no\nequivalent data are provided for the hybrids. This omission limits the strength of the\nclaim that resistance traits have been successfully transferred.\nThank you for pointing out this important issue. We have now included the disease\nresistance data for the hybrids and their maternal parents. These results have been\nintegrated into Table 1.\n• A new table should be added to report disease resistance data for the hybrids using\nthe same scoring or index system used in Table 1.\nThe hybrids were evaluated using the same scoring system as the parental lines,\nensuring methodological consistency. To facilitate comparison and maintain a concise\npresentation, the results were added to Table 1 rather than placed in a new table.\n• The “Disease Index” formula referenced in Table 1 should be added to the “Methods”\nsection at least, to ensure transparency and reproducibility.\nThe formula for calculating the “Disease Index” has been added to the Methods\nsection. Please see lines 663-665.\n• The use of “NS” to denote “no symptoms” is inconsistent across the table as NS also\nappear in the IP column.\nFor table 1, the “NS” in the IP column has been changed to “NA” for clarity. The\ntable note now reads: “NA: Not Applicable, as incubation period cannot be calculated\ndue to absence of symptoms.”\nMetabolite analysis and pigmentation\n• The authors report the detection of numerous low-abundance anthocyanins (Figure\nS18). However, the authors have not provided any detail in their methods to evaluate\nthe reliability of their compound identification or quantification. Critical information\nmissing includes sample preparation protocols, chromatographic separation parameters,\nMS/MS acquisition settings, and criteria used for compound identification. This lack of\ntransparency is particularly concerning given that several anthocyanins share identical\nm/z values, making chromatographic resolution and MS/MS spectral interpretation\nessential for accurate compound differentiation. For metabolite quantification the authors\nshould report whether they used any standards for quantification. Without this\ninformation, it is difficult to assess whether the low-abundance signals are from true\nanalytes or are analytical artifacts.\nThank you for pointing out this important issue. We have revised the Methods section\nto include additional details on metabolite identification and quantification (lines\n921-936).\n• Following this, the authors suggest that the black stem colouration is due to\naccumulation of specific anthocyanins presented in Supplementary figure 18b. Whilst\nthe authors have reported statistical significance in compound accumulation, given\ntheir extremely low abundance (for example 0.02 ug/g pelargonidin-3-O-arabinoside)\ncompared to other identified compounds (eg. in S18a procyanidin B2 is reported at\naround 300 ug/g for both M. cheesmanii and the green stemmed Musa acuminata) is\nthis difference likely to be biologically relevant in creating the black stem phenotype?\nThe authors should note that dark brown or black colouration can come from sources\nother than anthocyanins. Oxidation of phenolics which have two neighbouring\nhydroxyl groups can lead to the formation of quinone-like structure which can further\nundergo polymerisation making brown/black colouration. Examples include caffeic\nacid, catechol, catechin, dopamine and L-DOPA. Dopamine and L-DOPA have\npreviously been detected in various banana tissues at high concentrations.\nAdditionally, as the first 3 compounds share many of the genes/enzymes with the\nanthocyanin biosynthesis pathway, the authors should consider adjusting the\nmanuscript (including transcriptome analysis) to encompass all phenolics rather than\nonly anthocyanins.\nWe appreciate your insightful comment and fully agree that pigmentation is not solely\ndetermined by anthocyanins. In response, we have expanded our analysis to include\ngenes involved in the oxidative metabolism of phenolic compounds, which are known\nto contribute to dark or black pigmentation through enzymatic polymerization.\nSpecifically, we examined the expression of polyphenol oxidase (PPO), peroxidase\n(POD), and DOPA decarboxylase (DDC), key enzymes involved in these pathways\n(lines 418-420). However, our results showed no significant upregulation of these\ngenes in the black-pseudostem samples, suggesting that phenolic compounds may\ncontribute less to pseudostem pigmentation than anthocyanins. In addition,\nconsidering that metabolites with low abundance are less likely to contribute\nsignificantly to the black pseudostem coloration, we only selected the top 10 most\nabundant metabolites in the black pseudo-stem (Mchb) of M. cheesmanii for\ncomparison. Based on this analysis, we found that the delphinidin-based pathway may\nplay an important role in the black coloration of M. cheesmanii pseudostems, and we\nhave revised the corresponding results accordingly (lines 451-463).\nTranscriptomic validation (qPCR)\n• The claim that transcriptomic findings were validated by qPCR (lines 377-378) is\nmisleading. For example, the gene F3’H is not statistically significant based on\nSupplementary Figure 15a, and there is no presented qPCR data for PAL2 or\nBGLU26, even though these genes are mentioned.\nThank you for pointing out this important issue. We have revised the description of\nthe qPCR validation to ensure it accurately reflects the results. Specifically, we\nclarified that BGLU26 and PAL2 failed to amplify, and F3'H showed higher\nexpression in black pseudo-stems but not significantly. The revised text now provides\na more precise and detailed account of the differential expression analysis. Please\nrefer to lines 432-439.\n• The qPCR methodology is also not described. The manuscript should include details\nabout cDNA synthesis protocols, primer sequences, and amplification conditions in\nthe “Methods” section.\nThank you for this pointing this out. We have now added the relevant details on cDNA\nsynthesis protocols, primer sequences, and amplification conditions in the Methods\nsection (lines 909-920).\n• To support transparency and reproducibility, the authors should also consider\nproviding raw Ct values as a supplementary table.\nWe thank you for this suggestion. We have now provided the raw Ct values in Source\nData (sheet 5) to support transparency and reproducibility.\n• Supplementary Figure 8a appears to be nearly identical to Figure 4a in the main text.\nTo avoid redundancy, one of these figures should be removed.\nWe agree with your suggestion and have removed Supplementary Figure 8a to avoid\nredundancy with previous Figure 4a (now Figure 5a) in the main text.\nMinor revisions and editorial suggestions\n• The authors should ensure consistent terminology and formatting across all tables\nand figures, particularly for labels and abbreviations.\nWe have carefully checked all tables and figures to ensure consistency in terminology,\nlabels, and abbreviations throughout the manuscript.\n• All abbreviations should be defined upon first use in each table or figure, even if\nthey are explained elsewhere in the manuscript. For example, “eBSV” should be\nredefined as “endogenous banana streak virus” where it first appears.\nAll abbreviations have been checked and are now defined upon their first appearance\nin each table and figure.\nDiscussion\nThis is a good discussion supported by a solid foundation of relevant literature.\nCertain sections, such as the paragraph beginning at line 509, primarily summarise\nresults rather than examining their broader implications within the context of existing\nresearch.\nWe have revised the Discussion section by reducing descriptive result summaries and\nexpanding the interpretation of findings in the context of existing research.\nThe discussion does not consider some of the practical challenges associated with the\nuse of M. cheesmanii in breeding programs. These may include low hybrid recovery\nrates, the potential for linkage drag, and the complexity of backcrossing strategies.\nAdditionally, phrases such as “revolutionary breakthrough” and “remarkable\nproductivity” are used in the text without sufficient supporting evidence and do not\nalign with the otherwise analytical tone.\nWe appreciate your helpful suggestions. We have added a new subsection titled\n“Breeding challenges and future prospects for M. cheesmanii utilization” (lines\n597-619) to address the practical limitations. In addition, although we have included\ncomparative analyses of disease resistance, agronomic traits, and fruit quality between\nthe hybrids and their parents, we have also revised the wording throughout the\nmanuscript to more objectively reflect the results.\nExpanding the discussion to include resistance genes, specifically identifying the\ntypes of loci involved, could add value. Comparing these loci to those found in other\npublished Musa genomes, and considering their potential implications for breeding\nstrategies, would provide additional depth and relevance.\nYour suggestion is very helpful. We have added a genome-wide survey of resistance\ngenes and expanded the discussion accordingly (lines 339-361 and 551-560).\nThe structure of the discussion may be improved by the inclusion of subheadings to\nsupport clarity and assist reader navigation. Several paragraphs are lengthy and would\nbenefit from being divided into smaller units. Although the intended audience is likely\nto have technical expertise, certain terms could be clarified to increase accessibility.\nFor example, “eBSV” could be restated as “endogenous banana streak virus,” and\nterms such as “segregation distortion” and “structural heterozygosity” could be\naccompanied by short explanations.\nWe appreciate your suggestions on improving clarity. We have added subheadings,\nsplit lengthy paragraphs, and clarified terms. Regarding “segregation distortion” and\n“structural heterozygosity,” we removed the related sentences. Structural\nheterozygosity refers to differing structural configurations between homologous\nchromosomes within an individual (e.g., an inversion or translocation present on one\nhomolog but not the other), which can affect meiotic pairing and segregation. The\nstructural variations reported in our study are not heterozygous within individuals,\nand thus this concept is not applicable here.\nThe presentation of data may be strengthened through brief agronomic interpretation.\nFor instance, a girth to height ratio of 0.225 could be described as exceeding the\nthreshold associated with resistance to lodging.\nWe agree with your comment. To the best of our knowledge, no universally\nestablished threshold exists for the girth-to-height (G/H) ratio directly linked to\nlodging resistance in bananas, but a higher G/H ratio is generally advantageous\nbecause a thicker pseudostem relative to plant height improves structural stability and\nreduces lodging risk under environmental stresses such as strong winds or heavy fruit\nloads. We have strengthened this discussion in the revised manuscript (lines 585-588).\nFinally, the discussion could also benefit from consideration of future directions, such\nas whether similar traits may be found in other underutilised Musa species or what\nrisks could emerge from the widespread deployment of M. cheesmanii in breeding\nprograms.\nWe have expanded the discussion to include the potential applications and future\nprospects of M. cheesmanii in banana breeding, as suggested (lines 614-619).\nMethods\nCritical methodological details are missing in several areas, which limits the\nreproducibility and rigour of the study.\nThe description of Fusarium oxysporum f. sp. cubense TR4 inoculation lacks essential\ninformation. The origin of the fungal strain, its vegetative compatibility group (VCG),\nand the inoculation protocol (particularly the spore concentration and method of\napplication) are not provided. In addition, the criteria used to evaluate external and\ninternal symptoms of infection, and how disease severity was scored or calculated, are\nnot adequately described.\nThank you for pointing this important issue. In response, we have revised the\nMethods section to provide full details of VCG, inoculation protocol, and disease\nscoring system (Please see lines 643-644, 646-648, 660-665). The strain VCG01216\nwas tested. In the greenhouse, for each accession, fifteen plantlets were inoculated by\napplying 2 mL aliquots of spore suspension into five equidistant holes around the\nplant base near the rhizome, while control plants received sterile water. Both disease\nseverity scoring and disease index (DI) calculation were performed according to Li et\nal. (2015). Briefly, disease severity was evaluated using external symptoms and\nrhizome discoloration (internal symptoms) on a 0-3 scale, and the DI was calculated\nas [Σ(score in the scale × frequency)/(total number of plants × maximum class in the\nscale)] × 100. External symptoms were recorded every 2 days starting 7 days after\ninoculation, and internal symptoms were assessed upon plant death or at 65 days.\nField evaluations were conducted at an experimental plot in Nanning, Guangxi (22°48’\nN, 108°22’ E; Alt. 106 m) from 2023 to 2025 using 15 acclimatized plants per\naccession in a randomized block design. External symptoms were assessed every 2\nweeks, and rhizome discoloration was recorded when plants showed severe symptoms\nor at 21 months. These additions ensure that the inoculation procedure and disease\nassessment are clearly described and fully reproducible.\nLi, W. M., Dita, M., Wu, W., Hu, G. B., Xie, J. H., & Ge, X. J. (2015). Resistance\nsources to Fusarium oxysporum f. sp. cubense tropical race 4 in banana wild relatives.\nPlant Pathology, 64(5), 1061-1067.\nHybrid evaluation is similarly underdeveloped. Terms such as “seed set,” “seed\ngeneration,” and “normal seedling” (lines 592-593) are ambiguous and require precise\ndefinitions. The section describing hybrid assessment (lines 592-597) lists variables\nthat were evaluated but does not explain how these measurements were conducted.\nSample sizes for hybrid offspring are also not reported, and replication details are\nentirely missing.\nThank you for this comment. The term “seed set” has been defined as the average\nnumber of seeds produced per fruit (line 691). The term “normal seedling” has now\nbeen defined as “seedlings with healthy and regular appearance” (line 692). In\naddition, “seed generation percentage” has been corrected to “seed germination\npercentage” (line 691). And we have added essential measurement conditions as well\nas sample sizes for these traits in Table 2.\nThe anthocyanin analysis lacks transparency, with key methodological details missing.\nSpecific recommendations for improving this section are outlined in the Results\ncritique above.\nWe are grateful for your comment on this important aspect. We have updated the\nMethods section with expanded details on metabolite identification and quantification\n(lines 909-936).\nqPCR validation of transcriptomic data is not described at all. There is no information\nabout cDNA synthesis, primer sequences, qPCR reaction conditions, or normalisation\nstrategies. To improve transparency and reproducibility, the authors should include\nthese details in the methods and provide raw Ct values in a supplementary table.\nThank you for pointing out this omission. We have now added a detailed description\nof the qPCR validation of the transcriptomic data in the Methods section, including\ncDNA synthesis procedures, primer sequences, qPCR reaction conditions, and\nnormalisation strategies (see lines 909-920). In addition, the raw Ct values have been\nprovided in Source Data (sheet 5) for full transparency and reproducibility.\nFinally, a specific section on statistical analysis should be included.\nThank you for this suggestion. We have added a subsection titled “Statistics and\nreproducibility” in the Methods section to describe the statistical analyses used in this\nstudy (see lines 944-952).\nTables and figures\n• The overall quality and readability of the tables and figures is good. However,\nFigures 1 and 2 would benefit significantly from higher-resolution images and\nimproved formatting to enhance visual clarity.\nHigh-resolution versions of Figures 1 and 2 have been provided, and their formatting\nhas been improved to enhance visual clarity.\n• In Figure 2, scale bars are missing from all panels and should be added to ensure\ninterpretability, particularly for morphological comparisons.\nScale bars have been added to all panels in Figure 2.\n• In Table 2, the definition of “plant stature” is placed in the footnotes, should also be\nadded to the main text (Methods). Table 2 needs to include data from parental lines,\nplant height measurements, standard deviations, and the number of replicates. A new\ntable reporting hybrid disease resistance should be created, modelled on Table 1.\nThank you for this comment. We have now added the definition of “plant stature” to\nthe Methods section (lines 693-695). We have revised Table 2 to include the parental\nline data and plant height measurements, with all data now presenting standard\ndeviations and sample sizes. Additionally, we performed supplementary disease\nresistance experiments and, using the same scoring system as previously, reported the\nresistance data for previously unreported parental lines ('Jinyu' and 'Yulin') as well as\nthe hybrids ('Jinyu' × M. cheesmanii; 'Yulin' × M. cheesmanii). The resultes have been\nincorporated into the existing Table 1 for clear presentation and comparison.\n• Figure 4, panel (e), which presents RGA2 polymorphisms, requires clearer labelling\nto highlight the nonsense mutation (T→A at 3,687 bp).\nThe original Figure 4e (now Figure 5e) has been revised to highlight this mutation in\n3,687 bp.\n• Figure 4 and Supplementary Figure 8 present nearly identical phylogenetic trees.\nTo avoid redundancy, one of these should be removed.\nSupplementary Figure 8a has been removed; please refer to the current\nSupplementary Figure 13.\n• Figure legends throughout should be made more descriptive, especially when\nreferring to multi-panel figures.\nFigure legends have been carefully checked and revised to clearly present the figure\ninformation.\nEnd of report\nReviewer #4:\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nWe truly appreciate the time, expertise, and constructive feedback you and your\nco-reviewer have provided, which have been invaluable in improving the overall\nquality of our work."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Many thanks for the opportunity to review this revised manuscript.\nThe authors have addressed all my previous concerns and I think the manuscript makes an extremely valuable contribution to the future of banana breeding, as well as providing an abundance of multi-omic data that other researchers can draw upon.\nI particularly commend the authors for augmenting the manuscript and providing additional information - furthermore, their key points are more clearly articulated than in the original version.\nI have no further comments and I feel that that the manuscript is acceptable for publication.", "reviewer_2": "The authors addressed all my requests and comments. I just have one last point regarding the assembly quality. The authors ran BUSCO, KAT, and Merqury after each step of the assembly process, and, as observed in many other projects, polishing tends to lower the quality of the assembly (as shown by the Merqury results and also by the loss of one complete BUSCO gene). I think the authors should indicate in the manuscript (for example lines 746 and 760) that polishing appears to have reduced the quality of some loci in the assembly.", "reviewer_3": "Review of Manuscript ID NCOMMS-25-27107A by Dr Huang and colleagues.\nManuscript title: Going wild in banana breeding: the cross-compatibility and multi-omics of Musa cheesmanii\nGeneral comments\nThe authors have made a commendable effort and have substantially improved the manuscript, addressing many of the reviewers’ previous concerns. However, a few issues remain that should be revised before the manuscript can be considered for publication.\nMinor revisions:\n1. The newly included Supplementary Figure 1 illustrates the improved shelf life of Yulin compared to its offspring; however, the data presented are limited to peel discolouration. In the figure legend, the authors note that “Yulin begins to collapse externally, with the pulp turning sour and rotting. In contrast, Haijiao retains its robust shape and desirable flavour.” Similar to comments made on the original manuscript, these observations lack supporting quantitative data. The authors are encouraged to include simple, quantifiable indicators of shelf-life to strengthen their claims. Parameters such as titratable acidity, pulp firmness, total soluble solids, weight loss, or respiration rate are routinely used to assess postharvest quality and could be readily incorporated. These measurable attributes would provide a more objective basis for evaluating fruit longevity and quality over time.\n2. In addition, statements referring to “desirable flavour” should be supported by appropriate evidence, such as sensory evaluation or compositional profiling (for example, measurements of sugars, acids, or volatile compounds). Including these analyses would substantiate the claim and enhance the credibility of the reported quality improvements.\n3. The comparison of flavonoid metabolite profiles is presented in Supplementary Figure 22 and Figure 6c; however, M. acuminata data are not shown in the bar charts of Figure 6c. The authors state that M. acuminata was excluded because it belongs to “clade A and is more distantly related to M. cheesmanii”. This rationale seems insufficient. Furthermore, in their analysis, the authors state that 4 anthocyanins, rutinoside and glucoside derivatives of delphinidin and petunidin, account for the black phenotype of M cheesmanii based on higher accumulation in the black stem compared to green stem. However, M. acuminata, which lacks black stem colouration, clearly accumulates higher levels of these compounds as shown in Supplementary Figure 22. This suggests that these anthocyanins alone do not account for the black phenotype, which may instead be derived from polymeric or derivatives of these compounds. The authors should revise the text accordingly.\n4. Finally on LC-MS/MS, the authors have improved their methodology reporting. However, they state they “compare with authentic standards” (line 934) and quantify “using external standards” (line 935). The authors should explicitly list which authentic standards were used for compound quantification. Given that they use MRM data for quantitation and that ionisation efficiency (and hence detection) can vary greatly between compounds, accuracy of the reported concentrations depends on the availability of appropriate authentic standards. If standards for all compounds were not available, the authors should clarify this and present the results as relative comparisons rather than as absolute quantifications (Fig 6c).\n5. Be specific when referring to Supplementary Figures:\nExample: line 412 “…replicates (Supplementary Fig. 17)” should be (Supplementary Fig. 17a) same for line 422 should be (Supplementary Fig. 17b)\n6. Section 423 to 439, seven genes are described as being up regulated, two did not amplify so Supplementary Fig. 20 should only show five genes. I see that the expression of F3’5’H is presented in this Figure but not described in the text.\n7. Line 431-443: It is not clear what the 6 TFs mentioned in line 443 refer to. The sentence says “Notably, 6 (24%) of these TFs were…” which would refer to the 12 TFs previously mentioned, in that case it should be 50% not 25%.\nEnd of report.", "reviewer_4": "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."}, "author_response": "Response to reviewers\nReviewer #1:\nMany thanks for the opportunity to review this revised manuscript.\nThe authors have addressed all my previous concerns and I think the manuscript makes an extremely valuable contribution to the future of banana breeding, as well as providing an abundance of multi-omic data that other researchers can draw upon.\nI particularly commend the authors for augmenting the manuscript and providing additional information - furthermore, their key points are more clearly articulated than in the original version.\nI have no further comments and I feel that that the manuscript is acceptable for publication.\nWe sincerely thank you for your positive evaluation and supportive comments, and we appreciate the time you dedicated to reviewing our work.\nReviewer #2:\nThe authors addressed all my requests and comments. I just have one last point regarding the assembly quality. The authors ran BUSCO, KAT, and Merqury after each step of the assembly process, and, as observed in many other projects, polishing tends to lower the quality of the assembly (as shown by the Merqury results and also by the loss of one complete BUSCO gene). I think the authors should indicate in the manuscript (for example lines 746 and 760) that polishing appears to have reduced the quality of some loci in the assembly.\nThank you very much for your further comment. As suggested, we have added a clarification in the manuscript indicating that polishing appeared to slightly lower the quality of the assembly (line 784-786).\nReviewer #3:\nReview of Manuscript ID NCOMMS-25-27107A by Dr Huang and colleagues.\nManuscript title: Going wild in banana breeding: the cross-compatibility and multi-omics of Musa cheesmanii\nGeneral comments\nThe authors have made a commendable effort and have substantially improved the manuscript, addressing many of the reviewers’ previous concerns. However, a few issues remain that should be revised before the manuscript can be considered for publication.\nMinor revisions:\n1. The newly included Supplementary Figure 1 illustrates the improved shelf life of Yulin compared to its offspring; however, the data presented are limited to peel discolouration. In the figure legend, the authors note that “Yulin begins to collapse externally, with the pulp turning sour and rotting. In contrast, Haijiao retains its robust shape and desirable flavour.” Similar to comments made on the original manuscript, these observations lack supporting quantitative data. The authors are encouraged to include simple, quantifiable indicators of shelf-life to strengthen their claims. Parameters such as titratable acidity, pulp firmness, total soluble solids, weight loss, or respiration rate are routinely used to assess postharvest quality and could be readily incorporated. These measurable attributes would provide a more objective basis for evaluating fruit longevity and quality over time.\nWe thank you for the suggestion to include quantitative postharvest measurements. We agree that the previous description lacked sufficient quantitative support. As suggested, to complement the observed changes in peel discoloration, we conducted additional analyses of key postharvest quality parameters, including fruit weight loss, pulp firmness, and malondialdehyde content (lines 234-238, 733-739; Supplementary Table 3). These indicators provide quantitative support for the observed extension of shelf-life in 'Haijiao No. 1' compared with 'Yulin'.\n2. In addition, statements referring to “desirable flavour” should be supported by appropriate evidence, such as sensory evaluation or compositional profiling (for example, measurements of sugars, acids, or volatile compounds). Including these analyses would substantiate the claim and enhance the credibility of the reported quality improvements.\nWe thank you for bringing this point to our attention. We have conducted both sensory assessment and chemical composition analysis in the first round of revision (Supplementary Tables 1 and 2). We apologize for the oversight in not presenting these results in the main text. We have now added the following sentence to the manuscript (lines 229-234):\nFourth, sensory evaluation showed that the hybrid fruit scored significantly higher than its maternal ABB cultivar for overall acceptability, indicating greater preference (Supplementary Table 1), while chemical composition analysis revealed significantly higher levels of β-carotene, soluble sugars, starch, carbohydrates, and sucrose (Supplementary Table 2), suggesting enhanced nutritional value and improved sweetness and overall eating quality.\n3. The comparison of flavonoid metabolite profiles is presented in Supplementary Figure 22 and Figure 6c; however, M. acuminata data are not shown in the bar charts of Figure 6c. The authors state that M. acuminata was excluded because it belongs to “clade A and is more distantly related to M. cheesmanii”. This rationale seems insufficient. Furthermore, in their analysis, the authors state that 4 anthocyanins, rutinoside and glucoside derivatives of delphinidin and petunidin, account for the black phenotype of M cheesmanii based on higher accumulation in the black stem compared to green stem. However, M. acuminata, which lacks black stem colouration, clearly accumulates higher levels of these compounds as shown in Supplementary Figure 22. This suggests that these anthocyanins alone do not account for the black phenotype, which may instead be derived from polymeric or derivatives of these compounds. The authors should revise the text accordingly.\nThank you for this valuable comment. Indeed, plant pigment formation is highly complex, and attributing the higher accumulation of these compounds in M. acuminata solely to lineage differences may be overly simplistic. We have revised the Discussion to address this point and added the following text in lines 514-523:\nFinally, although four delphinidin-based compounds were more abundant in black than green pseudo-stems of clade B species, their levels were even higher in the clade A species M. acuminata, which also has green pseudo-stems (Supplementary Fig. 22). While this pattern may reflect lineage-specific coloration mechanisms, the complexity of pigment formation means that these identified metabolites alone may not fully explain the black phenotype. The coloration may instead arise from polymeric products, derivatives of these compounds, differences in the relative proportions of individual anthocyanins, or other factors. Therefore, although these metabolites are likely associated with pseudo-stem coloration in clade B species, further studies are needed to elucidate the underlying mechanisms across the Musa genus.\n4. Finally on LC-MS/MS, the authors have improved their methodology reporting. However, they state they “compare with authentic standards” (line 934) and quantify “using external standards” (line 935). The authors should explicitly list which authentic standards were used for compound quantification. Given that they use MRM data for quantitation and that ionisation efficiency (and hence detection) can vary greatly between compounds, accuracy of the reported concentrations depends on the availability of appropriate authentic standards. If standards for all compounds were not available, the authors should clarify this and present the results as relative comparisons rather than as absolute quantifications (Fig 6c).\nWe thank you for this important point. As suggested, we have added Supplementary Table 30 listing all standards used. In our targeted LC–MS/MS analysis, absolute quantification was performed using authentic external standards whenever available. Commercial standards of the major anthocyanidin aglycones, including 7 cyanidins, 5 delphinidins, 3 malvidins, 3 peonidins, 1 pelargonidin, 1 petunidin, 5 procyanidins, and 2 flavonoids, were used to generate individual calibration curves.\nFor complex glycosylated, acylated, or otherwise structurally modified anthocyanins for which no commercial standards exist, semi-quantification was carried out using the calibration curve of the corresponding aglycone (surrogate-standard approach), which is widely used in targeted MRM workflows and provides reliable relative-to-absolute estimates within each anthocyanidin class. Importantly, among the compounds highlighted in Fig. 6c, five out of the six had direct standard-based calibration.\nWe have revised the Methods section to explicitly list all standards used and clarify the calibration and quantification strategy (lines 960-964).\n5. Be specific when referring to Supplementary Figures:\nExample: line 412 “…replicates (Supplementary Fig. 17)” should be (Supplementary Fig. 17a) same for line 422 should be (Supplementary Fig. 17b)\nThank you for pointing this out. We have revised all Supplementary Fig.17 citations to indicate the corresponding subpanels. Other supplementary figure citations are checked and updated as needed.\n6. Section 423 to 439, seven genes are described as being up regulated, two did not amplify so Supplementary Fig. 20 should only show five genes. I see that the expression of F3’5’H is presented in this Figure but not described in the text.\nWe thank you for pointing this out. We agree that the expression of F3'5'H should have been described in the Results section. We have now added the corresponding description in lines 445-447 to ensure consistency with Supplementary Fig. 20.\n7. Line 431-443: It is not clear what the 6 TFs mentioned in line 443 refer to. The sentence says “Notably, 6 (24%) of these TFs were…” which would refer to the 12 TFs previously mentioned, in that case it should be 50% not 25%.\nWe thank you for pointing out this confusion. The 6 TFs referred to were identified from species-level comparisons (Mac vs. Mchb and Mba vs. Mchb), rather than from the 12 TFs mentioned earlier. The Mch vs. Mchb comparison was not included because both samples are from the same species and therefore does not provide meaningful information regarding species-level responses to environmental differences. Overall, a total of 37 TFs showed significant differential expression in the species-level comparisons, among which 6 were linked to light responses (approximately 16%). We have corrected the percentage and revised the text to clarify this point (lines 452-454).\nEnd of report.\nReviewer #4:\nI 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.\nThank you for your time and support."}, {"round": 3, "reviewer_comments": {"reviewer_3": "Thank you for the opportunity to review the revised version of this manuscript. This is my third round of review, and I am pleased with the changes made by the authors.\nThe authors have responded thoroughly to the points raised in earlier reviews, and the manuscript is now clearer, more cohesive, and better supported. In my view, the study provides an important contribution to banana improvement research, and the datasets presented will be a valuable resource for the community.\nI have no further comments and I recommend the manuscript for publication.", "reviewer_4": "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."}, "author_response": "Response to reviewers\nReviewer #3:\nThank you for the opportunity to review the revised version of this manuscript.\nThis is my third round of review, and I am pleased with the changes made by the authors.\nThe authors have responded thoroughly to the points raised in earlier reviews,\nand the manuscript is now clearer, more cohesive, and better supported. In my\nview, the study provides an important contribution to banana improvement\nresearch, and the datasets presented will be a valuable resource for the\ncommunity.\nI have no further comments and I recommend the manuscript for publication.\nWe sincerely thank you for your positive evaluation and supportive comments, and\nwe appreciate the time you dedicated to reviewing our work.\nReviewer #4:\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training\nin peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.\nThank you very much for your time and support."}]

Decision letter