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Whole genome sequencing of a banana wild relative Musa itinerans provides insights into lineage-specific diversification of the Musa genus

Crop wild relatives are valuable resources for future genetic improvement. Here, we report the de novo genome assembly of Musa itinerans, a disease-resistant wild banana relative in subtropical China. The assembled genome size was 462.1 Mb, covering 75.2% of the genome (615.2Mb) and containing 32, 456 predicted protein-coding genes. Since the approximate divergence around 5.8 million years ago, the genomes of Musa itinerans and Musa acuminata have shown conserved collinearity. Gene family expansions and contractions enrichment analysis revealed that some pathways were associated with phenotypic or physiological innovations. These include a transition from wood to herbaceous in the ancestral Musaceae, intensification of cold and drought tolerances, and reduced diseases resistance genes for subtropical marginally distributed Musa species. Prevalent purifying selection and transposed duplications were found to facilitate the diversification of NBS-encoding gene families for two Musa species. The population genome history analysis of M. itinerans revealed that the fluctuated population sizes were caused by the Pleistocene climate oscillations, and that the formation of Qiongzhou Strait might facilitate the population downsizing on the isolated Hainan Island about 10.3 Kya. The qualified assembly of the M. itinerans genome provides deep insights into the lineage-specific diversification and also valuable resources for future banana breeding.

In total, the assembly comprised 7194 scaffolds (above 2 kb) with N50 of 192 kb, and 103,534 contigs (above 100 bp) with N50 of 33.9 kb (Table 1, Supplementary Table 3). The post-assembly quality evaluations showed that the distribution of GC content was concentrated around 0.388, well in accord with that of the published genome M. acuminata 8 , and the average base coverage was about 92× ( Supplementary Figs 3 and 4). The total heterozygous rate was estimated to be 0.25% based on the ratio of the number of single-nucleotide polymorphisms to the overall genome length, after excluding 'N' sites of assembly gaps (Supplementary Note 4). Using the package CEGMA 15 , the complete and partial coverage of core eukaryotic gene sets (CEGMA) for the M. itinerans genome assembly were 79.8% and 94.4% respectively, which were very close to the M. acuminata genome assembly (81.9% and 93.6% respectively) (Supplementary Table 4). Thus, we obtained a qualified draft genome assembly, which should be valuable genomic resources to support banana breeding.
Using both homology-search against the RepBase library 16 , and de novo predictions with RepeatModeler approaches, 38.95% (179.99 Mbp) of the Musa itinerans genome were predicted to contain repetitive elements, somewhat higher than that of M. acuminata ( Table 9, Supplementary Fig. 7). A 5-way comparison of the three Musa species and two closely related Arecaceae species P. dactylifera, and E. guineensis showed that 7810 gene families were shared among them, and 2723 uniquely shared gene families between the three Musa genomes (Fig. 1a). M. itinerans shared more gene families with M. auminata (2727 gene families) than with M. balbisiana (140 gene families), which were suggestive of higher similarity for the latter two species. The unique gene families of M. itinerans among the five species were significantly enriched for 40 GO terms and involved in some essential functional categories such as DNA polymerase activity (GO: 0034061, P = 1.56e-9, hyper-geometric test), endoribonuclease activity (GO: 0016891, P = 5.12e-9, hyper-geometric test), and ribonuclease activity (GO:0004540, P = 4.15e-7, hyper-geometric test) (Supplementary Table 10). Twelve genes were significantly mapped on the ribosome pathway (ko03010, P = 4.64e-5, hyper-geometric test) (Supplementary Table 11), and it has been suggested that ribosomes might play some roles in the intrinsic regulation of a subset of mRNAs translation with unique cis-regulatory elements 17 . The followed proteasome pathway (ko03050, P = 1.77e-5, hyper-geometric test) involved the degradation of abnormal or damaged proteins in the cell, and played crucial roles in response to diverse intracellular signals and changing environmental conditions such infection, heat and cold etc 18 . Other enriched pathways fell into diverse categories with development process, growth, response to biotic or abiotic stresses, such as plant hormone signal transduction (ko04075), inositol phosphate metabolism (ko00562), fatty acid biosynthesis (ko00061), plant-pathogen interaction (ko04626), and starch and sucrose metabolism (ko00500) etc. (Supplementary Table S11). Overall, these unique gene families of M. itinerans might also give some hints on how a subtropical marginally distributed species coped with the challenging environmental conditions. Comparison of orthologous gene families among nine related species revealed that 2870 expanded and 1032 contracted gene families in the ancestor of the three Musa species (Fig. 1b). The expanded gene families were enriched in 29 pathways, of which the most striking expansions were associated with phagosome (ko04145, hyper-geometric test, P = 2.18e-20), and plant-pathogen interaction (ko04626, hyper-geometric test, P = 1.11e-19) (Supplementary Table 12). Phagosome biogenesis was a key process in tissue remodeling, clearing apoptotic cells, and restricting the spread of intracellular pathogens 19 , together with the plant-pathogen interaction pathway (ko04626), both of which provided some clues on how tropical emergent taxa coped with the intrinsic or exogenous biotic stresses. Besides these two important functional categories, the remaining 27 pathways involved in many other important component biogenesis processes, and might be associated with some phenotypic or physiological innovations in the ancestral Musa species (Supplementary Table 12). In contrast, the ancestral Musa species lost fewer gene families, and only 15 pathways were significantly over-represented (Supplementary Table 13). One noteworthy pathway, phenylalanine metabolism (ko00360, P = 3.44e-05, hyper-geometric test) was considered to be a key step in the lignin biosynthesis of vascular plants, and the contraction of this pathway might perturb the lignin biosynthesis and cell-wall architecture 20 , thus might result in the crucial transition from a woody to herbaceous disposition in the ancestors of Musa species. As one of the most marginally distributed Musa species in subtropical areas, 21 expanded and 24 contracted pathways were enriched for M. itinerans (Supplementary  Tables 14 and15), some of which were in accordance with the release from biotic-stresses such as pathogens (ABC transporter 21 , ko02010, P = 1.25e-17, hyper-geometric test; plant-pathogen interaction, ko04626, P = 2.28e-9, hyper-geometric test), and intensification of abiotic stresses such as drought and cold (phagosome 19 , ko04145, P = 6.61e-24, hyper-geometric test; amino sugar and nucleotide sugar metabolism 22 , ko00520, P = 8.13e-13, hyper-geometric test).  Table 16). The global view of the genome M. itinerans including gene density, GC content distribution, repeat elements distribution were also plotted along the pseudo-chromosomes (Fig. 2), and it showed that these genes unevenly distributed among given window slides (P = 0, Chi-square test), and positively correlated with the GC content significantly (P = 2.2e-16, Spear's rank correlation rho = 0.35), as predicted by GC content and gene density 24 . Using the MCScanX Program 25 , 510 syntenic blocks covered 36.25% of gene repertoire of M. itinerans were identified, with average number of 13, ranging from 6 to 164 (Fig. 2f); and 695 syntenic blocks composed of 36.10% of the final gene set in M. acuminata were found ( Supplementary Fig. 10). Based on the distributions of synonymous substitutions rate (Ks) of these syntenic orthologous and paralogous gene pairs, the speciation and paleoploidization events in the Musa genus were inferred. When following an average Ks of 3.47 per 10 9 years for nuclear genes in the Zingiberales 8 , M. itinerans and M. acuminata diverged approximately about 5.8 million years ago with peak Ks around 0.04 (Fig. 3). This divergence time estimation was consistent with that based on the phylogeny of 1201 single copy nuclear genes among nine plant species (Fig. 1b, Supplementary Fig. 8). For both Musa species, one peak around K s 0.40~0.42 for intragenomic syntenic paralogs indicated an ancient genome duplication event occurred about 57.6~61.0 Mya, which was consistent with the polyploidization explosions in many plant species around Cretaceous-Tertiary boundary 26 . According to the ancestral syntenic blocks inferences in Musa 8 , this genome duplication should correspond to the two in separate successive duplication events (α /β ) in M. acuminata.    Table 17). The decreasing number of NBS genes was consistent with the latitudinal ascending of the three species. During the transition from humid tropical to cool subtropical habitats, some NBS related genes would be less numerous due to relaxed selection constraints on them. The absence of Toll/interleukin-1 receptor (TIR)-like (TIR-NBS types) in the three Musa species was consistent with previous reports in monocots 27 , and these NBS genes were distributed unevenly among the 11 chromosomes with skewed distributions on chromosome 3, 6, 9, respectively ( Supplementary Fig. 11). A large proportion of the NBS-encoding genes were singletons, and only a few of them clustered into gene families (Supplementary Table 17, Supplementary Fig. 12). The predominant transposed duplication mode for the NBS-encoding genes might contribute to the widespread singletons ( Supplementary Tables 18 and 19), which was prone to cause drastic gene structural divergence and function diversification 28 . Transposed duplication modes were found to be responsible for the proliferation of other important gene families including MADS-box, F-box, B3 transcription factors in Brassicales 29 . In addition, transposed duplications were observed to span wider time scales than other duplication modes in both Musa species ( Supplementary Fig. 13). With duplication ages in terms of Ks ranged from 0.01 to 5.50 with average 1.59 for M. itinerans, and from 0.02 to 3.93 with average 0.83 for M. acuminata, vast majority of the NBS-encoding genes were duplicated well prior to the divergence of M. itinerans and M. acuminata (Ks = 0.04), hence, the ancient ages of these duplicated NBS-encoding genes were not in accordance with the classic arm-race model, which predicted a high turnover rate and predominant young NBS-encoding genes within a genome 30,31 . K a /K s (nonsynonymous to synonymous substitution ratio) values of most duplicated pairs (segmental duplication, tandem duplication, proximate duplication, transposed duplication) within both genomes were less than 1, indicating that purifying selection played an essential role in the maintenance of intra-genomic diversity of these NBS-encoding genes in Musa, as the cases in chestnut 32 , sorghum 33 , Solanum 34 . In our comparative studies in Musa, the NBS-encoding gene repertoire seemed to be associated with their specific habitat shift.
Transcription factor (TF) genes for the two Musa species were identified by blast against known transcription factor gene families of related species, as well as homology prediction using all the known transcription factor proteins to blast against the M. itinerans genome. In total, 3176 and 2898 putative transcription factor genes, from 58 families, were identified for M. itinerans and M. acuminata, respectively (Supplementary Table 20). These numbers represent 9.8% and 7.9% of the 32,456 and 36,542 predicted protein-coding loci. Among the 58 transcription factor families identified, MYB, AP2, Dof, G2-like, SBP, HD-zip, WOX, RAV, LBD and SAP were over-represented in both Musa species relative to other taxa ( Supplementary Fig. 14). Similarly to M. acuminata, the M. itinerans genome contains a large number of TFs that can be explained by the 3 rounds of specific WGD in the Zingiberales order 8 .
The MYB family was the largest transcription factor family in the two Musa species. With the number of repeats in the N-terminal conserved MYB DNA-binding domain, the MYB super-family can been categorized into four classes, 1R-, R2R3-, 3R-, and 4R-MYB, and the largest one R2R3 MYB was plant specific and key regulators in metabolism and responses to biotic and abiotic stress, also in flowering or fruit development 35 . About 292 and 304 MYB genes were identified in the genomes of M. acuminata and M. itinerans after manual inspection, respectively (Supplementary Table 21, Supplementary Fig. 15), and 92.1% and 93.4% of them were R2R3-type MYB genes, and much higher than in Arabidopsis (56.8%) and rice (70.1%) 36 . The extensive collinearity between M. itinerans and M. acuminata for these MYB gene pairs was observed (Supplementary Fig. 16a). The intragenomic duplicated MYB gene pairs also showed good synteny and relatively even distribution across chromosomes ( Supplementary Fig. 16b,c). For M. acuminata, this gene family arose primarily by segmental duplications (about 186 gene pairs) with median Ks = 0.57 and followed by transposed duplications with median Ks = 0.89 (about 97 gene pairs), both of which were prior to the ancient α /β whole genome duplication events (Ks = 0.40~0.42) in Musaceae (Supplementary Table 22). The MYB TFs' gene families are essential to plant morphogenesis and diverse physiological processes, and the gene balance hypothesis assumed that these regulating gene families were dosage sensitive and robust to gene loss after whole genome duplication events 37 .About 89.7% of these segmental duplicated gene pairs were under purifying selection (with K a /K s < 1), followed by transposed duplications (about 97 gene pairs, 84.6% of them with K a /K s < 1). Under this scenario, purifying selections were expected to wipe out deleterious mutations and stabilize the MYB TFs gene repertoire for their essential functions. Similar evolutionary patterns were observed in M. itinerans (Supplementary Table 23 and subsequent expansions were expected to reduce the effective population sizes, however, population migrations and concomitant admixtures during expansions among highly heterogeneous populations might lead to increased N e 42 on the other hand, the heterogeneous topography in South China would result in the persistent population substructure, which was also possible for the elevated Ne during the glacial and interglacial cycles. It's possible that the counter effect of admixtures between populations of M. itinerans overrode that of the population bottleneck until the progressive disappearance of population heterogeneities at the Last Glacial Maximum (LGM, c.a 18-20 Kya). However, due to genetic deficiency or less frequent admixtures, the isolated HN population showed more rapid decay of heterogeneity and earlier shrinkage of Ne than either of the continental populations (Fig. 4). The final formation of the Qiongzhou Strait was in the Middle Holocene when marine strata transgressed the original lowland, and the Hainan Island was completely isolated from Leizhou Peninsula 43 , so an additional Ne reduction of the HN Island population was observed at the time 10.3 Kya.

Conclusion
Genome sequencing of the wild banana Musa itinerans, one of the most disease-resistant wild banana species, provides invaluable genetic resources for future banana genetic improvement. The M. itinerans diverged from M. acuminata only 5.8 Mya, and exhibited extensive collinearity between them, but the two species occupy distinct habitats, which provides an unpreceded chance to unravel the diversification within the Musa genus. Resorting to lineage-specific gene family expansions and contractions approaches, a batch of candidate genes categorized into diverse pathways or GO terms were inferred to be associated with some peculiar traits. However, more delicate experiments were required to clarify their roles in the physiological or phenotypic innovations, as one of the most disease resistant and cold hardy species in the Musa genus, the gene repertoire space of M. itinerans should be mined in the future. The non-synonymous to synonymous ratio K a /K s of < 1 for most NBS-encoding gene family revealed pervasive purifying selections, together with the ancient duplication ages, both in support of the trench-warfare model hypothesis and against the classic arms-race model. A similar evolution pattern was also observed in the MYB transcription factor family, in which the purifying selection wiped out deleterious mutations and stabilize the MYB TFs' gene repertoire for their essential functions. Demographic history has a profound effect on the genetic diversity of extant species. The isolated HN population of M. itinerans was more prone to genetic loss during glacial and interglacial cycles than the counterpart mainland populations, in which admixture differentially buffered against genetic loss. Furthermore, the detailed scenarios of demographic history for this marginally distributed species will be revealed with more extensive sampling and population genome resequencing studies.

Methods
Sampling preparation and Sequencing. Natural populations of M. itinerans are distributed in subtropical China, and individual leaves of M. itinerans were harvested and immediately storied with sillica-gel in the field (the source of material see Supplementary Notes 1). The total genomic DNA was extracted with the CTAB (cetyl trimethylammonium bromide) method, and qualified with A260/280 ratio and agarose gel electrophoresis for further use. To obtain a high-quality reference genome, we sequenced one individual of M. itinerans from Hainan population using a whole-genome shotgun sequencing strategy. Paired and mate-pair Illumina genomic DNA libraries with varying insert size (180 bp, 500 bp, 2 kb, 5 kb, and 10 kb) were constructed following the manufacturer's instructions. All the libraries were sequenced on the Illumina Hiseq2000 platform. Raw reads were subject to the removals of PCR duplicates, adaptor sequences, and contaminants of bacterial or viruses. The processed high-quality reads were assembled into contigs and scaffolds using SOAPdenovo2 package 14 and gaps were filled using Gapcloser (version 1.12, http://soap.genomics.org.cn/soapdenovo.html). The reference genome size of M. itinerans was estimated based on the K-mer frequency distribution analysis (details for Supplementary Note 2). Two individuals each from the YC and LC populations were sampled and sequenced using a paired-end library size of 500 bp with > 30-fold coverage. Genome annotation. For the draft genome of M. itinerans, interspersed repeats were identified using RepeatMasker v3.30 (http://www.repeatmasker.org) and homolog research for the Repbase database 16 . Repeat proteins were characterized using RepeatProteinMask v3.30 with default parameters, de novo interspersed repeats were annotated using RepeatModeler (http://www.repeatmasker.org/RepeatModeler.html). Tandem repeats in the genome of M. itinerans were screened using Tandem Repeat Finder v4.04 44 . Next, a homology searching approach was used to identify protein-coding genes. We obtained the protein sequences of Musa acuminata v1 from the Banana Genome Hub 45 and Phoenix dactylifera DPV01, Oryza sativa IRGSP-1.0, Sorghum bicolor v3.1, Zea mays, and Arabidopsis thaliana TAIR10 from Phytozome (https://phytozome.jgi.doe.gov) or related databases (Supplementary Note 5) and then blasted against the repeat-masked genome assembly of M. itinerans using TBLASTN 46 with percentage identity over 70% and coverage not less than 50%. Gene models were predicted based on these alignments using Genewise 47 . De novo gene predictions were performed using Augustus 48 and GENSCAN 49 . Both sets of genes were integrated using GLEAN 50 . The annotations of the final gene sets were obtained by searching against the protein database KEGG 51 , SwissProt 52 , TrEMBL 53 using an e-value cut-off of 1e-5, and motifs and domains for the gene set were determined using InterProScan 54 . Using CEGMA package, the completeness of the gene space for the draft genome assembly was evaluated with the highly conserved 248 core eukaryotic gene (CEGs).
For noncoding RNAs, tRNAs were identified using tRNAscan-SE 55 , snRNAs and miRNAs, and rRNAs were obtained by searching the genome assembly against the Rfam database using InFERNAL with default parameters (http://infernal.janelia.org/). All the raw short reads, genome assembly were deposited in NBCI's Sequence Read Archive (SRA) database with accession number PRJNA312694, and the gene annotation used for the analyses were provided to the Banana Genome Hub (banana-genome-hub.southgreen.fr/organism/Musa_itinerans).

Genome-wide duplications and estimation of the insertion times for LTR retrotransposons.
Using the web-based tool SynMap on the plant comparative genome platform CoGe (https://genomevolution. org/CoGe/) 56 , the syntenic regions between M. itinerans and M. acuminata were generated, and scaffolds or contigs of M. itinerans were mapped or oriented on the 11 pseudo-chromosomes of M. acuminata. Reciprocal BLASTP between protein sequences of two Musa species with E-value cut-off of 1e-5, were implemented to identify orthologs between species, and self BLASTP for both species were used to obtain paralogs within species. After removing the self-matches of the BLASTP results, the syntenic blocks (≥ 5 genes per block) were determined with the package MCScanX 25 . The remaining aligned results were used to generate dot plots. Paralogous gene pairs in each block within species were used to infer genome duplication events. Meanwhile, orthologous gene pairs in each block between species provided some insights into the speciation events. The four-fold transversion (4DTv) ratio for each gene pair in the block was calculated on concatenated nucleotide alignments with HKY substitution models, and the distributions of these 4DTv values were used to estimate the whole genome duplication events and speciation events.
The 5′ -and 3′ -LTR sequences of the full-length LTR retrotransposons were aligned, and the K value (the average number of substitutions per aligned site) were calculated in the package MEGA 57 . The insertion times were estimated using this formula: T = K/(2 × r), where r represents the average substitution rate and is estimated to be 1.30 × 10 −8 substitutions per site per year in rice 39 .
Phylogenetic analysis. The protein sequences of eight plant species including Arabidopsis thaliana, Vitis vinifera, Oryza sativa, Sorghum bicolor, Phoenix dactylifera, Elaeis guineensis, M. acuminata, and M. balbisiana were downloaded (sources details in Supplementary Note 5). After filtering out protein sequences with amino acid length of less than 50, the longest protein sequence was chosen among spliced variants. All the eight protein datasets together with those of M. itinerans were clustered into paralogous and orthologous groups using the program OrthoMCL v1.4 with the inflation parameter of 1.5 58 . Protein sequences of each single-copy gene obtained from the nine species were aligned using MUSCLE v3.8.31 59 , and corresponding nucleotide sequences alignment of different genes were concatenated into one supergene. Using 4DTv sites of the supergene with the nucleotide substitution model HKY85 + gamma, a maximum likelihood phylogenetic tree of the nine species was constructed using the package PhyML with NNI (Nearest Neighbor Interchange) tree improvement 60 . Bootstrap supports of the internal branches were evaluated by 1000 full-heuristic searches. Divergence time estimations between species were determined using MCMCtree in PAML 4.7 61 , and the "correlated molecular clock" and "REV" substitution model were used in the approximate likelihood calculation. The MCMC process was run for 2,000,000 steps and sampled every 20,000 steps. A calibrated divergence time of 124~248. 4 Ma between Arabidopsis and Sorghum was used 23 . Gene family expansions and contractions of extant species and ancient lineages were estimated using CAFE v2.1 62 . Using a random birth and death model, the gene birth (λ ) and death (μ = − λ ) rates were estimated across the species tree composed of M. itinerans and eight other species, using the maximum likelihood method, and gene families with accelerated rate of expansion and contraction were determined with a threshold conditional P-value (P < 0.05).
Gene family evolution. Using HMMER V3 (http://hmmer.janelia.org/software), protein sequences of M. acuminata, M. itinerans, and M. balbisiana were aligned against the raw hidden Markov model (HMM) corresponding to the Pfam NBS (NB-ARC) family (PF00931, downloaded from website http://pfam.sanger.ac.uk/). Following the method described in D' Hont et al. 8 , the hit NBS-domain sequences were aligned and used to construct a new NBS HMM profile using the module 'hmmbuild' . Using this Musa specific model, we identified NBS-candidate proteins in the three Musa species respectively. TIR and LRR domains of these NBS-encoding amino acid sequences were identified with Pfam_scan. Coiled-Coil (CC) motif was identified using MARCOIL 63 with a threshold probability of 90 and double-checked using paircoil 64 . Duplication modes of these NBS-encoding genes were identified in MCScanX 25 .
Scientific RepoRts | 6:31586 | DOI: 10.1038/srep31586 Transcription factors' gene families (TFs) of the M. itinerans were identified by two complementary approaches. First, protein sequences of M. itinerans were BLASTP against the TFs of five other species including Musa acuminata, Arabidopsis thaliana, Vitis vinifera, Oryza sativa, Phoenix dactylifera (http://planttfdb.cbi.pku. edu.cn/). Second, de novo predicted TFs were obtained using all the TFs of the other five species to TBLASTN against the genome of M. itinerans and corresponding gene models were predicted using Genewise 47 . Using GLEAN 50 , non-overlapping TFs of M. itinerans were obtained. The duplication history of the MYB gene family was inferred using the programme MCScanX 25 .
Demographic history inference. One individual from each of the three populations was sequenced in high depth (Supplementary Note 1), and qualified reads from the short insert size library (500 bp or 180 bp) were realigned to the assembly with BWA with default parameters 65 . Consensus sequences were called using samtools 66 , and converted into the Fastq format using bcftools and vcfutils in the package pairwise sequentially Markovian coalescent model 38 (PMSC). The consensus sequences were split into non-overlapping 100 bp bins marked as homozygous or heterozygous and these split sequences were used to reconstruct the demographic history with the PSMC model with the following parameters: "N30 -t15 -r3 -b -p "4 + 5 * 3 + 4". The variance of the simulation results was assessed with 100 bootstrap replicates. Finally, the PSMC profiles were scaled using generation time (g) of one year, and neutral mutation rate μ of 1.30 × 10 −8 substitution per site per year.