Origin of small chromosome A08 and genome evolution of Arachis species

Domain: neuroscience
Published: 2026-01-26
Editorial outcome: major_revision

Abstract
Wild Arachis species exhibit abundant genetic diversity for peanut improvement. However, the evolutionary history of their genomes is unclear. Here, through comparative oligopainting, we establish a one-to-one correspondence between microscopically observed chromosomes and sequenced pseudomolecules of Arachis duranensis, A. ipaensis, and cultivated peanut, and determine the 10 homoeologous groups (Hgs) of the A, B, F, K, and H genomes. Analysis of the telomere-to-telomere (T2T) genome assembly of A. hoehnei reveals that its genome is a diverging form of the A genome and is designated as genome A’. In addition, the unique small chromosome A08 is originated from two inversions and a substantial contraction of A'08. We propose a genome evolution model of the Arachis genus, where A’ bridges the A and B genomes. We further report an artificial hexaploid peanut derived from a hybrid of cultivated peanut and A. hoehnei, and identify differentially expressed genes against web blotch in A. hoehnei.

Introduction
Peanut (Arachis hypogaea L., AABB, 2n = 4x = 40), an important food and oil crop, is an allotetraploid and self-pollinating crop with a narrow genetic background. Arachis species, some of which have excellent resistance to pests, diseases, and drought, represent an invaluable gene pool for peanut breeding1. Several genes conferring resistance to nematodes, rust and leaf spot have been introgressed from wild species into cultivated peanut2–4.

The Arachis genus diverged from other legumes approximately 50 million years ago (Mya)5. The genus originated in South America and is widely distributed from the eastern foothills of the Andes in Bolivia and northern Argentina to the Atlantic coast in Brazil and from the southern limit of the Amazon region to the northern coast of the Rio de la Plata in Uruguay6,7. Arachis comprises 83 species8, which have been classified into nine sections based on their morphology, cross-compatibility, hybrid viability, geographic distribution, and cytogenetic characteristics, namely, Trierectoides, Erectoides, Procumbentes, Rhizomatosae, Heteranthae, Caulorrhizae, Extranervosae, Triseminatae, and Arachis9–11. Section Arachis includes cultivated peanut, the tetraploid wild species A. monticola, and 29 diploid wild species. Currently, the genomes of Arachis are preliminarily divided into the A, B, D, F, K, and G genomes12–17. However, clear evidence supporting the origin and genome evolution of these species is still lacking.

Genome sequencing facilitates an understanding of the origin and genome evolution of Arachis species. The A genome of A. duranensis and the B genome of A. ipaensis were considered the donors of cultivated peanut, which diverged approximately 2–2.60 Mya18. Species with the A genome harbor a smaller size chromosome A08, also called “small chromosome” or “A chromosome”19. Comparative genomics analyses validated the genomic origin of cultivated peanut, where high collinearity of between wild species and cultivated peanut exists in both A and B genomes. The small chromosome A08 in A-genome species was probably derived from translocations and inversions between chromosomes 7 and 8 of its ancient ancestor, where the repeat-enriched segment of chromosome 8 was translocated to chromosome 7, while the gene-enriched segment of chromosome 7 was translocated to chromosome 820. To date, genome assemblies of Arachis species have been reached to telomere-to-telomere (T2T) level21–24. Recent pangenome analyses of eight Arachis species and cultivars have revealed frequent structural variations (SVs) during peanut evolution, and 1335 SVs likely associated with domestication have been identified in the A and B genomes25.

Existing studies mainly focused on the A and B genomes of the Arachis species. Limited information is available for D, F, G, H, and K genomes, e.g. genome identity and evolution, homoeologous chromosome pairs or groups. Comparative oligopainting using chromosome-specific single-copy oligonucleotide probe libraries (CSCOLs) and tandem repeat oligonucleotides provides a fast and efficient tool for determining homoeologous chromosome pairs and large segmental structural chromosome variations (SCVs) during the genome evolution of different species in large samples26,27. In addition, the rapid development of T2T genome assembly techniques facilitates the accurate identification of whole-genome variations28.

In this work, comparative oligopainting not only reveals the correspondence of all the pseudomolecules of A. duranensis to its actual chromosomes but also determines 10 homoeologous groups (Hgs) and SCVs in the A, B, F, K, and H genomes. By combining this information with the T2T genome assembly of A. hoehnei (Zw61), we identify its genome identity, propose the origin of the small chromosome, and elucidate the genome evolution of Arachis species. Further, we report an artificial hexaploid peanut line derived from a hybrid of cultivated peanut and A. hoehnei, and identify differentially expressed genes against web blotch in A. hoehnei.

Results
Development of a consensus karyotype of A. duranensis
Based on the reference genome sequence of A. duranensis, 10 CSCOLs named L1A-1 to L10A-1 were designed for one or two terminal regions of chromosomes A01–A10. For each library, 26,365–27,392 unique oligonucleotides (42–48 nt in length) were designed from segments of 4–9 Mb (Fig. 1a and Supplementary Table 1). The oligonucleotides of each library were then mapped in silico onto the corresponding pseudomolecules of A. duranensis, and all sequences were consistent with the target regions (Supplementary Fig. 1).

L1A-1, L2A-1, L3A-1, L8A-1 and L9A-1 were labeled with digoxigenin-11-dUTP and detected using anti-digoxigenin-rhodamine, whereas L4A-1, L5A-1, L6A-1, L7A-1 and L10A-1 were labeled with biotin-16-dUTP and detected using a fluorescein-labeled antibiotin antibody. The CSCOLs were divided into five pairs, each containing one digoxigenin-11-UTP-labeled library and one biotin-16-dUTP-labeled library. We used these libraries to paint the chromosomes of A. duranensis (PI219823). As expected, all 10 CSCOLs produced clear signals in one or two terminal regions of a single pair of chromosomes corresponding to A01–A10 (Supplementary Fig. 2).

Sequential FISH was conducted using single-strand oligonucleotide (SSON) multiplex #1 (DP-1, DP-2, DP-3, DP-4, DP-5, DP-6, and DP-7) and 45S and 5S rDNA probes following CSCOL painting to determine the correspondence between the pseudomolecules and actual chromosomes of A. duranensis (Fig. 1b, Supplementary Table 2). A karyotype integrating signals from CSCOLs, SSONs and rDNA probes revealed their correspondence to chromosomes and pseudomolecules. The 10 chromosomes were clearly differentiated based on their unique signal patterns and were designated Adu01 through Adu10 (Figs. 1c, d). Analysis of the arm ratios of the actual chromosomes revealed that the six pseudomolecules started from the ends of the short arms of chromosomes Adu02, Adu04, Adu06, Adu07, Adu08 and Adu09, whereas the other four pseudomolecules were from the ends of the long arms of Adu01, Adu03, Adu05 and Adu10.

Each chromosome exhibited a unique signal pattern. Adu01 presented strong green signals for SSON in both the centromeric and short arm terminal regions. Adu02 exhibited very strong green SSON signals in the centromeric and pericentric regions. Adu03 presented only a very weak green telomere signal on its short arm. Adu04 showed strong green SSON signals in the centromeric and pericentric regions. Adu05 exhibited red and green signals in the centromeric and pericentric regions, as well as green signals from the telomeric repeats of DP-5 (a SSON probe) centrally along the long arm. Adu06 presented very strong red and green SSON signals in the centromeric and pericentric regions, along with 5S rDNA sites in the short-arm pericentric region. Adu07 is the only submetacentric (sm) chromosome in A. duranensis and is characterized by very strong SSON signals and red 45S rDNA signals in the centromeric and pericentric regions. Adu08 is the smallest chromosome in A. duranensis, with strong red and green SSON signals in the centromeric and pericentric regions. Adu09 has a very weak green SSON signal in the centromeric region. Adu10 showed weak green SSON signals in the centromeric region and strong 45S rDNA signals in the long-arm pericentric regions (Figs. 1c, d).

Determination of homoeologous chromosome pairs in Arachis species
The method described above was used to analyze 16 other Arachis species/accessions, including three A. hypogaea cultivars, A. monticola, and 12 wild diploid species/accessions, where A. duranensis (PI219823) was the control (Table 1, Supplementary Figs. 3 to 18). CSCOL painting produced clear signals in all species, allowing consensus karyotypes and ideograms to be established. This not only enabled identification of 10 homoeologous groups (Hgs) named Hg1–10 for Arachis species but also facilitated detection of SCVs that occurred during evolution of Arachis (Figs. 2 and 3a, Supplementary Table 3).

Each of the 10 CSCOLs produced clear signals on one pair of chromosomes in all 17 species/accessions, indicating that these unique single-copy sequence blocks are conserved. However, only three CSCOLs (L1A-1, L3A-1, and L4A-1) produced signals at similar positions on the same chromosome pairs in all species. The other seven CSCOLs either hybridized to different positions on the same chromosome pair or to different chromosomes entirely in the B subgenomes of A. hypogaea and A. monticola, as well as in A. ipaensis, A. magna, A. trinitensis, A. hoehnei, A. batizocoi, A. pusilla, and A. dardani. These findings indicate that large segmental SCVs occurred during Arachis evolution. A total of six SCVs were identified in these species, including four types of translocations, one inversion, and one more complex variation (translocation plus inversion) (Fig. 3b, c; Supplementary Fig. 19; Supplementary Table 3).

Among the SCVs, four translocations—between Hpu06 and Hpu10, between Hda05 and Hda09, between Hda02 and Hda10, and between Hda06 and Hda10—occurred in H-genome species (Fig. 3c, Supplementary Fig. 19). The inversion and complex variation involved an inversion of chromosome 8 of A. hoehnei relative to the “small chromosome” A08 and translocations between chromosomes 7 and 8 in the A. hoehnei genome relative to the B, F, K, and H genomes. Based on the variations in chromosomes 7 and 8, the 13 wild diploid species were divided into three groups: 1) Group I - pretranslocation types: it includes all species with the B, F, K, and H genomes, do not exhibit chromosomal translocations involving chromosomes 7/8. 2) Group II - posttranslocation but preinversion type for chromosome 8: includes only A. hoehnei. This species contains a subtelocentric chromosome 8 and a metacentric chromosome 7 formed by translocation between ancestral chromosomes 7 and 8. 3) Group III - postinversion type for chromosome 8: includes all A-genome species. These species are characterized by the small chromosome A08, which is inverted relative to chromosome 8 of A. hoehnei (Fig. 3b).

Additionally, L1A-1 and L3A-1 have extra signals in the pericentric regions of different chromosomes in three cultivars (Tifrunner TIF, Baisha1016 BS and Silihong SLH) and six wild species (A. monticola PI 263393, A. duranensis PI 468202, A. villosa PI 298636, A. herzogii PI 476008, A. simpsonii Grif14534 and A. diogoi PI 276235), indicating that some unique oligonucleotide sequences in A. duranensis were amplified in other genomes (Supplementary Figs. 3–11).

Cytological characterization of SCVs caused by tandem repeats
Sequential FISH using seven tandem repeat-derived SSON probes was conducted after CSCOL painting in 17 species/accessions to further clarify SCVs during evolution (Figs. 2 and 3a). A consensus karyotype integrating both the SSON and CSCOL signals was established. The karyotypes of the Ahy and Amo subgenomes of cultivated peanut and A. monticola were highly similar to that of the Adu genome of A. duranensis (PI219823 and PI468202). The karyotypes of the Bhy and Bmo subgenomes of cultivated peanut and A. monticola were nearly identical to those of the Bip genome of A. ipaensis (PI468322) and Bma genome of A. magna (PI 468337), except that chromosome Bma08 lacked 45S rDNA sites. In contrast, the karyotypes of the Av, Ahe, Asi, Adi, Kba, Ftr, Hda, and Hpu genomes, as well as the A. hoehnei genome, differed significantly from those of the A and B subgenomes of cultivated peanut and A. monticola (Figs. 2 and 3a).

The seven tandem repeat-derived SSON probes produced unique signals at different positions on the chromosomes: DP-1 signals were consistent with those of the 5S rDNA probe; probes DP-2, DP-3, DP-4, DP-6, and DP-7 produced signals in the centromeric and pericentromeric regions; and DP-5 produced signals in telomeric regions29. A comparison of the karyotypes revealed that chromosomes in the 10 Hgs exhibited variable copy numbers and presence/absence variations (CNVs and PAVs) associated with these seven tandem repeats. Heterogeneous sites of 45S rDNA and SSONs were frequently observed on chromosomes Hg1, Hg8, and Hg10 of Av; Hg6 and Hg10 of Ahe; Hg2, Hg4, Hg6, and Hg7 of Asi; and Hg2, Hg4, Hg5, and Hg8 of Adi. In particular, interstitial telomere repeats (ITRs) occurring at various positions, including telomeric, centromeric and interstitial chromosomal regions, were detected in almost all species (Figs. 2 and 3a).

The signal pattern and intensity variation in the 10 Hgs were as follows:
Hg1—chromosomes in this group displayed green ITR signals on the short arm in seven genomes (Ahy, Amo, Adu, Av, Ahe, Asi, and A. hoehnei genomes). 45S rDNA sites were detected only in the Av genome, whereas 5S rDNA sites were exclusive to the Kba genome. Green SSON signals were observed in the centromeric and pericentromeric regions of seven genomes (Ahy, Amo, Adu, Av, Ahe, Kba and Hpu), with SSON PAV signals detected in the Av genome. Yellow SSON signals were detected in eight genomes (Asi, Adi, Bhy, Bmo, Bip, Bma, Ftr and A. hoehnei genomes), with no signal in the Hda genome.

Hg2—this group contained 45S rDNA sites in seven genomes (Av, Ahe, Asi, Adi, Kba, Hda and A. hoehnei genomes) and 5S rDNA sites in the Kba genome. Both PAV and CNV (signal intensity variations) were observed, with PAVs occurring in the Asi and Adi genomes, and CNVs detected in the Av genome. Yellow SSON signals were observed in centromeric regions of Hg2 in all B and K genomes; red SSON signals were detected in the A. hoehnei genome; and green SSON signals were detected in the Asi and Adu genomes; and no signal was detected in the Hda genome. Additionally, this chromosome in the Av genome had telomeric signals. SSON PAVs were observed in the Asi genome.

Hg3—centromeric regions of this group showed strong red SSON signals in all B, K and F genomes; weak red signals in the Ahy, Amo and Adu genomes; green SSON signals in the Av, Ahe, Adi and Hpu genomes; and yellow SSON signals in the Kba genome. CNVs were detected in the Adi genome, but no centromeric signals were detected in the Asi and Hda genomes. Additionally, strong green ITR signals were also present on the short arms of Hg3 in the Kba and Hpu genomes.

Hg4—green centromeric SSON signals were detected in all A genomes; yellow SSON signals were detected in all B genomes; red signals were detected in the Ftr genome; and no centromeric signals were detected in the Kba genome. 45S rDNA sites were present only in the Hpu genome. In the Hda genome, Hg4 chromosomes exhibited green ITR and SSON signals in the centromeric and pericentric regions.

Hg5—no signal was observed on this chromosome group in the B, F, and H genomes. The Kba genome showed strong green centromeric SSON signals, whereas all the A genomes and A. hoehnei displayed yellow centromeric SSON signals. Five genomes (Ahy, Amo, Adu, Av and A. hoehnei genomes) had green ITR signals in the middle of the long arm.

Hg6—5S rDNA sites were present in all genomes except Hpu. The Bhy, Bmo and Bip genomes also contained 45S rDNA sites. Centromeric SSON signals were green in the A. hoehnei and Hpu genomes but were yellow in all other genomes.

Hg7—chromosomes in this group were submetacentric (sm) in all A genomes but metacentric (m) in other genomes. 45S rDNA sites were detected in all genomes except H and A. hoehnei. Centromeric signal patterns included strong red SSON signals in A. duranensis PI468202, B and F genomes; yellow SSON signals in K and A. hoehnei; strong green signals in the A genome of A. duranensis PI 219823 and A. simpsonii; and weak red or green signals in the other genomes.

Hg8—the unique “small chromosome” A08 of all A-genome species displayed yellow centromeric SSON signals. In the A. hoehnei genome, Hg8 chromosomes were subtelocentric (st) with strong yellow centromeric SSON signals. Red centromeric SSON signals were predominant in all B genomes. 45S rDNA sites were present in the Bhy, Bmo, Bip and Hpu genomes, and the Hpu genome additionally contained 5S rDNA sites.

Hg9 — extensive green ITR signals were observed in the centromeric regions of these chromosomes in the B and F genomes. The short arms of Hg9 chromosomes in the Hpu genome also had green ITR signals, but no centromeric signal was detected in any A genome except Ahe.

Hg10 — 45S rDNA signals were detected in all the A genomes except Av. The A. hoehnei genome contained both 45S rDNA and 5S rDNA sites, whereas no rDNA signal was detected in the B, F, K and H genomes. Centromeric signals were green in the A and K genomes, yellow in the B and A. hoehnei genomes, and absent in the H genome. Large telomeric signals were detected at chromosome ends in the Av and Asi genomes.

Telomere-to-telomere genome assembly of A. hoehnei
Cytological analysis revealed that four types of large segmental SCVs (translocations, inversions, CNVs and PAVs) occurred during evolution. Most of these variations are distributed in tandem repeat-enriched regions, including telomeres, centromeres and nucleolar organizer regions (NORs). Based on the evolution of the small chromosome, the A. hoehnei genome seems to be an evolutionary bridge between the B and A genomes. The genomes of A. duranensis and A. ipaensis were clearly designated and well assembled20, but the genome of A. hoehnei remained unknown (Fig. 4a). Therefore, a T2T genome assembly of A. hoehnei and comparative genomic analysis should not only validate these SCVs but also provide a more precise landscape of the origin of the “small chromosome” and genome evolution of peanut.

Genome sequencing resulted in ~67× coverage from raw ultralong Oxford Nanopore Technologies (ONT) data, ~80× coverage from PacBio High Fidelity (HiFi) data, ~49× coverage from Illumina reads, and ~96× coverage from high-throughput chromosome conformation capture (Hi-C) datasets for assembling the A. hoehnei genome (Supplementary Tables 4–6). Based on k-mer analysis of Illumina short reads, the genome size of A. hoehnei was estimated to be ~1.49 Gb, with a heterozygosity rate of ~0.37% (Supplementary Fig. 20a). Hybrid assembly of high-quality HiFi reads and ultralong ONT reads using Hifiasm (v0.19.9) and Verkko (v2.2.1) software to assemble the genome generated 149 contigs with an N50 size of 96.40 Mb. Contaminating sequences were identified by BLAST analysis against bacterial nt databases and organelle sequences and then removed, leaving 37 contigs with an N50 size of 96.39 Mb. Hi-C data were used to map the chromosomes, resulting in a draft A. hoehnei assembly that included 10 pseudomolecules with 20 gaps. These gaps were subsequently filled with HiFi data using QuarTeT software. The ONT data were further assembled using Flye software, and the remaining gaps were filled using TGS-GapCloser and Minimax2 software. A complete T2T assembly of all 10 chromosomes of the A. hoehnei genome was obtained, comprising a total size of 1609.1 Mb (Table 2 and Supplementary Table 7). The basic structural features (gene density, repeat sequence density, and GC density) of the A. hoehnei T2T genome are shown in Fig. 4b.

Multiple methods were used to evaluate the integrity and continuity of the genome. Hi-C interaction heatmaps validated the chromosome order and orientation, revealing strong interactions near the diagonal of each chromosome block, indicating the high-quality assembly of all 10 chromosomes (Fig. 4c). Illumina short reads, HiFi reads, and ultralong ONT reads were mapped to the T2T genome with mapping rates of 99.52%, 99.98%, and 97.23%, respectively. The quality of the T2T genome assembly further assessed using the BUSCO (Benchmarking Universal Single-Copy Orthologs) tool detected 99.13% of the core conserved genes, including 1502 single-copy genes and 98 duplicated genes (Supplementary Fig. 20b). The integrity of long terminal repeat (LTR) sequences was evaluated by calculating the LTR Assembly Index (LAI), which was 27.13, indicating a high-quality genome. A genome consensus quality value (QV) of 49.10 was obtained using Illumina data and the k-mer clustering method in Merqury, confirming that the assembly was highly complete. According to RepeatMasker software, 83.63% (1345.62 Mb) of the A. hoehnei genome consists of repetitive sequences, with transposable elements (TE) accounting for 71.98% (Supplementary Table 8) and tandem repeats accounting for 11.65% (Supplementary Table 9). LTR retrotransposons were the most abundant, comprising 65.02% of the genome, among which Gypsy (45.16%) and Copia (2.81%) were the most prevalent types (Supplementary Table 8).

Telomeres, centromeres, and NORs are key structural components of chromosomes. Using TIDK and FindTelomeres software, we identified 20 telomeric repeat segments on the 10 chromosomes. The telomeres ranged in length from 1004 to 11,265 bp, with an average length of 5721 bp (Supplementary Fig. 20c, Supplementary Table 10). The shortest telomere is located on the short arm of the subtelocentric (st) chromosome 8, and the longest is on chromosome 10. Additionally, the ITR signals from the telomeric probe DP-5 (TTTAGGG)8 were strong in the middle of the short arm of chromosome 1, medium in the middle of the long arm of chromosome 5, and weak near the centromere of chromosome 6. The copy numbers of DP-5 on these chromosomes were 17,546, 5624, and 1262, respectively, indicating that the signal strength increased with increasing copy number (Supplementary Fig. 21a). A BLAST search of the 500-bp subtelomeric sequences near the telomeres of the 10 chromosomes of A. hoehnei against the ITR sequences on chromosomes 1, 5, and 6 showed that these ITRs originated primarily from the telomeres at the starting position of chromosome 3, accounting for 75.22% (45,969 copies), 69.26% (14,919 copies) and 74.75% (3449 copies) of all ITR copies in chromosomes 1, 5 and 6, respectively, followed by the telomeres at the starting position of chromosome 6, accounting for 24.75%, 30.42% and 25.25% of all ITR copies in chromosomes 1, 5 and 6, respectively (Supplementary Table 11).

Centromere positions were predicted using Centromics, quarTeT, and CentIER software. The lengths of the 10 centromeres ranged from ~1.50 Mb to ~11.60 Mb, with an average length of 5.30 Mb. Chromosome 9 has the largest centromere, and chromosome 5 has the smallest, indicating substantial variation in centromere length across the A. hoehnei genome (Supplementary Fig. 21b). In terms of arm ratio, all chromosomes are metacentric (m) except 8, which is subtelocentric (st) (Supplementary Table 12), consistent with FISH karyotyping results. Further analysis of the centromeric sequences revealed a large number of short dispersed repetitive sequences (SINEs) in the centromeric region of chromosome 5, whereas the centromeric regions of the other chromosomes were rich in unknown long terminal repeat sequences (LTRs). Chromosomes 1 and 6 contain no coding genes in the centromeric regions. Among the chromosomes, chromosome 2 has the most coding genes (22) in the centromeric region. The other chromosomes have fewer coding genes in their centromeric regions (17 on chromosome 10, 9 on chromosome 9, 5 on chromosomes 8 and 5, 4 on chromosomes 4, 3 on chromosome 7, and 1 on chromosome 3) (Supplementary Table 12).

We sequenced the transcriptomes of roots, stems, leaves, flowers, and fruits; annotated the protein-coding genes of the A. hoehnei genome; used three prediction methods (homologous protein prediction, de novo prediction, and transcriptome prediction); and ultimately identified 46,625 genes (Supplementary Tables 13 and 14). The average gene length was 3510.12 bp, the average coding sequence (CDS) size was 1059.09 bp, and the average number of exons per gene was 5.06, with an average exon length of 1288.01 bp (Supplementary Table 15 and Supplementary Fig. 22). We also identified 10411 rRNA genes, 909 tRNA genes, 49 miRNAs, 50 snRNAs, and 79 snoRNAs across the genome. The 5S rRNA was transcribed from 5S rDNA, whereas the 18S, 5.8S, and 25S rRNAs were generated through splicing of the single 45S transcript encoded by the 45S rDNA. There were high-frequency 5S rDNA copies on chromosomes 2 (1581 copies, near 46 Mb), 6 (512 copies, near 39 Mb), and 10 (1,504 copies, near 79 Mb; and 3824 copies, near 136 Mb) concentrated near the centromeres and on long arms. In parallel, 5S rDNA FISH signals were strong on chromosome 10, weaker on chromosome 2, and weakest on chromosome 6. 45S rDNA was located mainly on chromosome 2 (566 copies, near 71 Mb; and 31 copies, near 96 Mb), chromosome 10 (390 copies, near 88 Mb), and chromosome 7 (12 copies) (Supplementary Table 16). FISH signals for 45S rDNA were strong on chromosomes 2 and 10, which are high-copy-number locations, whereas no signals were detected for the 31 copies on chromosome 2 and 12 copies on chromosome 7 (Supplementary Fig. 21a). These results show that the signal strength increased with copy number. In silico mapping using the seven tandem repeat-derived SSONs and 45S and 5S rDNA sequences further validated the high quality of the T2T genome assembly, where signals and even signal intensity in target regions corresponded with those in the actual chromosomes and T2T pseudomolecules (Supplementary Fig. 21a). However, some signals in actual chromosomes failed to correspond to their sequences in the pseudomolecules of A. duranensis, A. ipaensis, and cultivar Tifrunner (Supplementary Fig. 23), where five sites in A. duranensis and A. ipaensis, respectively, and two sites in Tifrunner showed clear signals that did not correspond to the probe sequences mapped in the pseudomolecules, thus indicating poor genome assembly of those regions.

Comparative genomic analysis
Eight Arachis species have either chromosome-level or T2T genome assemblies, including A. ipaensis, A. duranensis, A. cardensii, A. stenosperma, A. hypogaea (cultivars Tifrunner, YZ9102 and Shitouqi STQ), and A. monticola. These species, together with Arabidopsis thaliana, Cicer arietinum, Medicago truncatula and Glycine max, were used for analysis of divergence time of A. hoehnei. A phylogenetic analysis based on 390 single-copy genes using A. thaliana, C. arietinum, M. truncatula, and G. max as outgroup species indicates that Arachis species diverged from other leguminous species approximately 64.34 (50.01 – 89.47) Mya. The divergence between the B genome and the clade containing the A and A. hoehnei genomes occurred approximately 2.29 (2.23 – 2.35) Mya, and the A and A. hoehnei genomes diverged approximately 1.78 (1.654 – 1.90) Mya (Fig. 4d). These findings indicate that the A. hoehnei genome is more closely related to the A genome than to the B genome.

Chloroplast assembly and phylogenetic analysis were conducted to validate these results. Using the above outgroup species, phylogenetic analysis showed that Arachis species clustered into a single branch, which diverged into three lineages. Among the 19 species/accessions, two H-genome species formed lineage III, and the remaining 17 species were divided into two major lineages. Lineage II includes species with the Ftr, Bip, Bma, and Kba genomes, with the Ftr genome located at the base branch of the phylogenetic tree, indicating that it originated earlier than the Kba, Bip, and Bma genomes. Lineage I was further divided into two clades: cultivated peanut and two accessions of A. duranensis (PI 468202 and PI 219823) grouped as Clade A, and species with the Av, Ahe, Asi, and Adi genomes and A. hoehnei grouped as Clade B. This suggested that the A. hoehnei genome is closely related to the A genome (Fig. 4e), consistent with results described above.

Using the T2T genome of A. hoehnei as a reference, the whole-genome SNPs of 15 wild diploid species were identified and used for phylogenetic analysis. The species again clustered into three lineages consistent with the chloroplast results, whereby the A. hoehnei genome was closer to the A genome but diverged earlier (Supplementary Fig. 24). Therefore, its genome was designated A’.

Gene family clustering analysis of 12 species revealed 4,398 shared gene families; 367 gene families (1019 genes) were specific to A. hoehnei, a number far greater than that for specific gene families in the A (A. duranensis, 147 families, 354 genes) and B (A. ipaensis, 228 families, 538 genes) genomes (Fig. 5a). These specific genes were distributed across all 10 chromosomes of A. hoehnei, with chromosome 5 having the most, followed by chromosomes 3 and 10. The results of copy number analysis for all gene families across the 12 species are shown in Fig. 5b. Compared with cultivated peanut, A. duranensis and A. ipaensis, A. hoehnei has more multicopy genes, suggesting it underwent more gene duplication events during evolution. In addition, 1697 gene families were expanded, and 948 gene families were contracted (Supplementary Table 17). Sixty-five genes were positively selected during evolution of A. hoehnei. Functional enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that specific genes of A. hoehnei were enriched in pathways such as ether lipid metabolism, glycerophospholipid metabolism, base excision repair, ubiquinone and other terpenoid-quinone biosynthesis, aminoacyl-tRNA biosynthesis, purine metabolism and endocytosis (Supplementary Fig. 25, Supplementary Table 17). Some of the expanded genes and positively selected genes were significantly enriched in pathways such as nucleotide excision repair, DNA replication, mismatch repair, and homologous recombination (Supplementary Figs. 26 and 27). The expanded genes involved in these pathways in A. hoehnei indicate that this species evolved an enhanced DNA repair system to maintain its genome stability.

The T2T genomes of A. hoehnei and cultivar YZ9102 were used as references for comparison with other genomes to elucidate syntenic blocks and structural and sequence variations in the eight Arachis species. Based on the percentage of syntenic block length relative to genome size of each species, the average percentages of syntenic blocks across the three genomes representing these eight species were ordered as follows: B–B (84.01%) > A–A (67.11%) > A’–A (23.56%) > A–B (18.08%) > A’–B (17.45%). This order was consistent with previous phylogenetic analysis results and divergence times of the species (Fig. 4d, Supplementary Fig. 28, and Supplementary Data 1). However, frequent chromosomal rearrangements, including translocations, inversions, duplications, insertions and deletions (>50 bp), and sequence variations (SNPs and indels) were observed. Compared with the three reference T2T genomes (A’, YZ9102A and YZ9102B), the number of SVs (>50 bp) among the different genomes was ordered as A’–A (47,591) > A–B (30,955) > A’–B (28,717) > B–B (23,979) > A–A (22,836) (Supplementary Fig. 29, Supplementary Data 1). The numbers of SNPs and indels (<50 bp) were ordered as A’–A (4,317,609) > A–A (3682,424) > A’–B (2,122,313) > A–B (1,994,344) > B–B (1,069,269) (Supplementary Fig. 29, Supplementary Data 1). Compared with the A genomes, the A’ genome had inversions exceeding 5 Mb on chromosomes 1, 3, 5, 8, 9 and 10, whereas compared with all B genomes, the A’ genome had inversions exceeding 5 Mb on chromosomes 1, 5, 6, 7 and 9. Large segmental translocations or complex variations were also observed in chromosomes 7 and 8 (Supplementary Fig. 28). These results indicate that A. hoehnei is quite distinct from species containing both the A and B genomes.

The origin of the small chromosome
Chromosome A08 is the smallest and most obvious chromosome in the A genome15. The length of chromosome A08 in A. duranensis, A. cardensii, A. stenosperma, Tifrunner A, YZ9102 A, STQ A and A. monticola A ranged from 47.73 Mb to 60.52 Mb, with an average of 53.51 Mb. The length of chromosome B08 in Tifrunner B, YZ9102 B, STQ B, A. ipaensis, and A. monticola B ranged from 129.60 Mb to 137.84 Mb, with an average of 135.36 Mb. The length of chromosome A'08 was 115.65 Mb. The average number of genes on chromosome A08 was 3,073, that on chromosome B08 was 3580, and that on chromosome A'08 was 4,131 (Supplementary Table 18). Both cytological and comparative genomic analyses showed that chromosome A08 originated from three major rearrangements. First, segmental translocations between chromosomes A'07 and A'08 in A. hoehnei relative to the B genomes (Fig. 5c); second, two inversions in chromosome A08 relative to chromosome A'08; and third, one substantial genome contraction of chromosome A08 compared with chromosome A'08 (Fig. 5d, Supplementary Fig. 30, Supplementary Table 19).

Collinearity analysis was performed using the T2T genome sequences of YZ9102 and A. hoehnei to precisely determine the rearrangements and relative breakpoints. First, compared with the B genome of YZ9102, there were six major rearrangements in chromosomes 7 and 8 of A. hoehnei. These rearrangements include a segment from 28.50–113.00 Mb of chromosome B08 translocated to two regions (32.00–55.00 Mb and 91.00–101.00 Mb) in chromosome A'07 (Translocation 1 and 2 in Fig. 5d); a segment from the 0.03–1.41 Mb region of chromosome B08 translocated and inverted to the 101.05–102.49 Mb region in chromosome A'07 (Translocation–inversion 2 in Fig. 5d); a segment from the 107.69–122.44 Mb region of chromosome B07 translocated to the 64.48 – 80.96 Mb region in chromosome A'08 (Translocation 3 in Fig. 5d); a segment of 123.96–140.30 Mb of chromosome B07 translocated and inverted to the 85.99–103.62 Mb region of chromosome A'08 (Translocation–inversion 1 in Fig. 5d); and an inversion involving the 29.19 – 102.40 Mb region of chromosome B07 and the 55.85–90.76 Mb region of chromosome A'07 (Inversion 3 in Fig. 5d).

Second, compared with chromosome A'07, no large SVs were observed on chromosome A07, indicating a strong syntenic relationship between the two chromosomes and an evolutionary connection between the A’ and A genomes. However, compared with chromosome A'08, two large segmental inversions were present in chromosome A08. Inversion 1 was located in the 2.59 – 62.24 Mb region of chromosome A'08, whereas Inversion 2 was identified in the 64.50–80.96 Mb region. The breakpoints of Inversion 1 were at 229,789 bp and 4,934,848 bp on chromosome A08, and the breakpoints of Inversion 2 were at 5,708,618 bp and 20,016,695 bp. No breakpoint was located within a gene, but they were all in intergenic regions and involved LTR and TIR sequences. The difference in genome size between chromosomes A'08 (115.65 Mb) and A08 (60.52 Mb) was 55.13 Mb. In Inversion 1, both gene and TE contraction were observed. Specifically, the length of Inversion 1 on chromosome A'08 was 59.65 Mb, but on chromosome A08, it was reduced to only 4.70 Mb - a substantial reduction of approximately 54.95 Mb comprising the loss of approximately 46.35 Mb of TEs along with 495 genes, which have homologous genes in chromosomes B07 or B08 (Fig. 5d, Supplementary Fig. 30, Supplementary Table 19). KEGG enrichment analysis indicates that genes missing from chromosome A08 relative to chromosome A'08 were involved in various pathways, such as homologous recombination, carbon metabolism, nucleotide excision repair, mismatch repair, carbon fixation by the Calvin cycle, and DNA replication (Supplementary Fig. 31). Thus, the chromosomal deletions associated with Inversion 1 are a primary factor contributing to the observed disparity in chromosome lengths between chromosomes A'08 and A08. The 495 genes lost in Inversion 1 of chromosome A08 remained in chromosomes B07 and B08, suggesting that chromosome A'08 originated from translocations between chromosomes 7 and 8, followed by inversion and deletion events that led to the formation of A08.

Overall, the B, A’ and A genomes have undergone significant chromosomal rearrangement events, including large fragment translocations, inversions, and deletions. Chromosomal fragments enriched with repetitive sequences were transferred to chromosomes A'07 and A07, while gene-enriched fragments were transferred to chromosomes A'08 and A08, ultimately leading to the formation of the current small chromosome A08.

TE variations during genome evolution of Arachis species
Both genome survey and assembly indicate that the A’ genome is the largest among the available genomes of Arachis species that mainly contain the A and B genomes (Supplementary Table 20). Analyses of intact TEs in the T2T genomes of YZ9102 (AABB) and A. hoehnei revealed that the A’ genome had the most intact TEs (38,749), followed by the B (28,444) and A (20,689) genomes (Fig. 5e). Compared with YZ9102 A and B genomes, SVs involving intact TE insertions accounted for 28.85% and 24% of the total SVs, respectively (Fig. 5f). Intact TE insertions in genes or neighboring regions can cause differential gene expression. Among the three genomes, the A’ genome had the most intact TE insertions in genes (476) or neighboring regions, followed by the YZ9102 B (199) and YZ9102 A (106) subgenomes. These genes were enriched in similar GO terms (Supplementary Fig. 32). The genome size of YZ9102 A is 1.15 Gb, with 839 Mb of TE sequences, accounting for 72.90% of the genome. In contrast, the A’ genome contained 1159 Mb of total TE sequences, accounting for 71.98% of its genome (Supplementary Table 21). The TE length of the A’ genome was 320 Mb larger than that of YZ9102 A, accounting for most of the difference in size between the two genomes. Although the total TE length of the A’ genome was similar to that of YZ9102 B, the numbers of each intact TE type differed; the A’ genome contained more LTRs, whereas the YZ9102 B genome had more TIRs (Fig. 5e).

SSON FISH was performed on the chromosomes of Tifrunner and A. hoehnei using four oligonucleotide probes derived from the LTR/Gypsy sequences of the A and B genomes to validate the TE differentiation among the A, B and A’ genomes. Oligo A-4 and Oligo A-7 specifically painted A-genome chromosomes but not B-genome chromosomes, whereas Oligo A-9 and Oligo A-10 painted B-genome chromosomes but not A-genome chromosomes. For the A’ chromosomes, Oligo A-4 and Oligo A-7 produced signals, whereas Oligo A-9 and Oligo A-10 produced almost no signal (Fig. 5g). The distribution frequency of the four probes in the genome of A. hoehnei was similar to that in the A subgenome of YZ9102, further confirming that its A’ genome is closely related to the A genome (Supplementary Table 22 and Fig. 5g).

Interspecies hybridization and oligo GISH confirm A’–A genome proximity
The crossability and pollen fertility of hybrids reveal genome affinity of distant species. The pollen viability of a hybrid (named W1612) between cultivar Yuhua15 and A. hoehnei was 10.76%, and no pods were produced. Chromosomes of the hybrid were doubled by colchicine treatment, resulting in a synthetic hexaploid, designated Am1612. Analysis of simple sequence repeat (SSR) markers C41 and F115, confirmed by multiplex #1 FISH, revealed that Am1612 had 60 chromosomes, comprising chromosomes from the Ahy and Bhy genomes of Yuhua 15 and the A’ genome of A. hoehnei (Supplementary Figs. 33 and 34). Am1612 had significantly increased pollen viability (66.77%) (Fig. 6a), but considerably lower levels observed in cultivated peanut. The height of the main stem, number of branches, leaf length and width on the main stem, petiole length, number of pods per plant, and seed length in Am1612 were significantly lower than for Yuhua15 (Fig. 6b, Supplementary Table 23). The low pollen fertility and seed setting suggested chromosomal instability among the A’, Ahy and Bhy genomes. Oligo GISH analysis with green probes specific to A-genome repetitive sequences and red probes specific to B-genome repetitive sequences showed that 40 chromosomes of Am1612 displayed green signals, confirming the genomic composition of Am1612 and further suggested that the A’ genome shares a closer genetic relationship with the A subgenome of cultivated peanut (Fig. 6c).

Differentially expressed genes of A. hoehnei against web blotch
An evaluation of disease resistance revealed that A. hoehnei exhibited high resistance to web blotch (Supplementary Fig. 35a). Transcriptomic analyses identified differentially expressed genes (DEGs) across all periods post inoculation, comprising 233 upregulated DEGs (upregulated DEGs) and 146 downregulated DEGs (downregulated DEGs) (Supplementary Fig. 35b). KEGG enrichment analysis of the upregulated DEGs revealed pathways related to flavonoid biosynthesis, tropane, piperidine and pyridine alkaloid biosynthesis, plant-pathogen interactions, biosynthesis of secondary, and isoflavonoid biosynthesis. In contrast, the KEGG enrichment results for downregulated DEGs revealed pathways related to other types of O-glycan biosynthesis, plant hormone signal transduction, metabolic pathways, and starch and sucrose metabolism (Supplementary Fig. 35c and d). Among all the identified DEGs, 26 up- and 20 downregulated genes were implicated in SVs when compared to those from the A and B genomes. Among them, three genes related to plant hormone signal transduction, plant–pathogen interactions and MAPK signaling pathways (Supplementary Table 24) likely contribute to disease resistance mechanisms previously described in cultivated peanut30. Additionally, a pathogenesis-related protein (PR10) gene, Ast01G035260.1 (Supplementary Table 24), might play an important role in web blotch resistance, as reported in other crops31,32. In the A’ genome, this gene contained a 399 bp insertion compared with its homologs in the A and B genomes of YZ9102 (Supplementary Fig. 35e and f).

Methods
Plant materials
This study utilized 18 Arachis accessions, including 4 tetraploid peanut cultivars, 1 tetraploid wild species (A. monticola) and 13 diploid Arachis accessions (Table 1), which were all provided by the Henan Academy of Agricultural Sciences.

Interspecies hybridization and chromosome doubling
Interspecies hybridization of Yuhua15 and A. hoehnei was performed in a Yuanyang experimental field. F1 (W1612) pods and embryos were cultured on 1/2 MS medium supplemented with 3% sucrose. The seedlings underwent chromosome doubling (MS + 0.1 mg L–1 NAA + 0.4 mg L–1 6-BA + 5% sucrose + 0.05% colchicine for 12 days) and recovery culture (MS + 0.8 mg L−1 NAA + 3% sucrose), yielding synthetic polyploid Am1612. The pollen viabilities of W1612 and Am1612 were observed.

Bioinformatic pipeline for CSCOL design and labeling
Ten CSCOLs were developed from 12 target regions at the distal end of each of chromosomes Adu01 to Adu10. The methods followed Han et al71. and Du et al29. and were based on the reference genome of A. duranensis20 in PeanutBase (https://peanutbase.org/). Briefly, after excluding repetitive sequences with RepeatMasker, the sequences in the target region were divided into SSONs of 42–48 nt. Each single-copy oligonucleotide was aligned to the reference sequence of A. duranensis using the BLAST tool72 and screened for homologs with >75% similarity. Single-copy SSONs with dTm >10°C (dTm = Tm – hairpin Tm) were retained. Ultimately, approximately 27,000 single-copy oligonucleotides in each chromosome were selected to construct 10 libraries (Supplementary Table 1). The libraries were then synthesized by MYcroarray (Ann Arbor, MI, USA), amplified, and labeled with biotin-16-dUTP or digoxigenin-11-dUTP73.

Sequential FISH and karyotyping analysis
Chromosomes were prepared according to the protocol reported by Du et al29. Briefly, healthy lateral root tips were excised and treated with 2 mM 8-hydroxyquinoline at 25°C for 3.5 h. The root tips were then fixed in a 3:1 (v/v) solution of absolute ethanol: glacial acetic acid for 1 h. For chromosome slide preparation, the meristematic tissue of the root tip was placed onto slides, squashed in 45% glacial acetic acid, and then frozen at -80°C for 12 h.

Clones of wheat 5S and 45S rDNA were labeled with biotin-16-dUTP or digoxigenin-11-dUTP (Roche) by nick translation and detected with a fluorescein-conjugated antibiotin antibody or anti-digoxigenin-rhodamine antibody (Roche). Multiplex #1, which included TAMRA (6-carboxytetramethylrhodamine)-modified oligonucleotides DP-1, DP-3, DP-4, and DP-6 and the FAM (6-carboxyfluorescein)-modified oligonucleotides DP-2, DP-5 and DP-7, was obtained from Du et al29. Multiplex #A and multiplex #B were obtained from Du et al74. All oligonucleotides were synthesized and modified by General Biosystems Company (Chuzhou, Anhui).

FISH was performed on chromosome spreads using CSCOLs, followed by the removal of chromosome signals. Next, GISH was performed using multiplex #A and multiplex #B as probes to identify A and B subgenome chromosomes, respectively74. Subsequent FISH procedures using repetitive multiplex #1 (SSONs) and rDNA probes were then performed on the same slides to develop the karyotypes. A fluorescence microscope (Leica DM6000, Germany) was used to detect signals. Images were optimized for contrast and brightness using Adobe Photoshop.

Investigation, statistics and reproducibility
Five mature plants of Am1612 and its cultivated parent were selected for investigation and analysis of eight phenotypic traits, including height of the main stem, length of the first primary branch, number of branches, number of pods per plant, length and width of the top 3rd and 4th leaves on the main stem, petiole length, and seed length (10 seeds). Statistical analyses of the eight trait values were performed using Excel. Prior to difference analysis, a one-tailed F test (95% confidence interval) was conducted on each dataset to verify the homogeneity of variance. Based on the results of the F test, a one-tailed t test (95% confidence interval) was applied to determine differences between Am1612 and its cultivated parent.

For GISH and FISH analyses, three independent biological replicates were established. In each replicate, 3–5 cells were observed to verify the consistency of the signals. For karyotyping analysis, 3–5 cells of each accession were observed and analyzed. Chromosome size was measured in at least three metaphase cells of each species using Image-Pro Plus 6.0. Excel was used to calculate three key karyotypic parameters: arm ratio (long arm length/short arm length), individual chromosome length, and karyotype length. Arm ratio was used to classify the chromosomes of each species as metacentric (m, 1.00–1.70), submetacentric (sm, 1.71–3.00) or subtelocentric (st, 3.01–7.00).

For RNA-seq analyses of infected plants, third leaves of A. hoehnei at the 6–8-leaf stage were sampled at 0 (mock-inoculated control), 1, 3, 5, 7, and 14 days after inoculation with the causative pathogen Phoma arachidicola (strain YY187)30. Three biological replicates were analyzed at each sampling time point. Transcriptome sequencing and subsequent bioinformatics analyses were performed at BGI (Beijing Genomics Institute). RT‒qPCR (with the same RNA) was performed using a PrimeScript™ II Kit (TaKaRa, Dalian) and PowerUp SYBR Master Mix (Applied Biosystems, CA, USA), with ADH3 used as the reference gene (2−ΔΔCt method; 3 biological replicates/4 technical replicates).

Analysis of molecular markers
Codominant SSR markers C41 (F: CACGAGAAGCAGAGGAAGAAGA; R: GCAGCAACAACTCAGGGAAGT) and F115 (F: GAGGGATTCGCTCCACAG; R: CACTCCAATAATTCAAACA) were used to confirm the interspecies hybrid plants. The amplified products were separated by capillary electrophoresis on an Applied Biosystems 3500/3500 XL system. The data were analyzed, and polymorphic markers were screened using Genographer software (https://sourceforge.net/projects/genographer/).

Probe mapping
The sequences of the multiplex #1 (SSON) rDNA probes exhibiting >80% homology were identified in the reference genomes of A. duranensis, A. ipaensis, A. hoehnei, and Tifrunner by BLAST searches (http://mcgb.uestc.edu.cn/b2dsc). Genome positions with more than 50 matches of DP-1, DP-2, DP-3, DP-4, DP-5, DP-6, and DP-7 or more than 1 match of 5S rDNA or 45S rDNA per Mb were mapped.

Genome sequencing, assembly and assessment of A. hoehnei
The A. hoehnei genome was sequenced using the PacBio HiFi, ONT ultralong, Hi-C, and Illumina NovaSeq 6000 systems at BGI. The ONT ultralong and HiFi reads were combined and assembled using Hifiasm (v0.19.9)75 and Verkko (v2.2.1)76, with the optimal assembly selected. Contaminants (mitochondrial, chloroplast, and bacterial sequences) were removed by BLAST alignment. The contigs within each chromosome group were ordered and oriented, and the interactions between contigs were transformed into a specified binary file using 3D-DNA77 and Juicer78. Juicebox78 was used to visualize and guide the manual sequencing and orientation of the contigs. First, HiFi sequencing data and quarTeT79 software were used to preliminarily fill the gap region. Second, ONT ultralong sequencing data were assembled using Flye, and the gap region was filled with TGS-GapCloser80 and Minimap2. Finally, NextPolish281 was used for error correction to obtain the final genome assembly.

BUSCO11 was used to evaluate completeness and integrity of the assembled A. hoehnei genomes and the uniformity of the sequencing coverage. Sequencing data were mapped onto the genome using BWA and Minimap2. Consistency quality values (QVs) of the assembled genomes were assessed with Merqury82. LAIs of the assembled genomes were determined using repetitive sequences83. Potential telomere repeat units in the genome were identified using TIDK (https://github.com/tolkit/telomeric-identifier). Potential centromere sequences and their positions were identified using third-generation reads and Centromics (https://github.com/JanaSperschneider/FindTelomeres).

Genome annotation and comparative genomic analysis
De novo prediction of the repetitive sequence database of A. hoehnei was performed using RepeatModeler284, and the database was then used to predict TEs in the genome with RepeatMasker85. Tandem repeats were identified mainly using Microsatellite86 and Tandem RepeatFinder87, with all predicted repetitive sequences subsequently integrated.

For gene coding predictions, the root, stem, leaf, flower, fruit needle, fruit and other tissues of the plant were sampled for transcriptomic sequencing, with three biological replicates included for each sampling group. Three prediction strategies were applied: de novo prediction using Augustus88 and SNAP and homologous species-based prediction using GeMoMa. These predictions were integrated using EVM, modified with PASA, and annotated against databases such as NR, eggNOG, GO, KEGG, TrEMBL, KOG, SWISS-PROT, and Pfam.

Circlize software was used to generate circle plots. Minimap2 was used to align the A. hoehnei genome with previously published genomes, and SyRI identified syntenic regions, structural rearrangements, and sequence variations. Centromere regions were analyzed using StainedGlass. Orthoender v2.4 and the PANTHER V15 database enabled gene family identification and annotation. GO and KEGG enrichment analyses were performed for species-specific gene families, and gene copy numbers were analyzed within each family across species.

MAFFT was used to align each individual single-copy gene sequence, IQ-TREE’s ModelFinder was used to select optimal models, and maximum likelihood (ML) was used to construct an evolutionary tree. The divergence time was calculated using MCMCTREE with fossil calibration times retrieved from the TimeTree website (http://www.timetree.org/). CAFE (v4.2.1) software was used to analyse gene family contraction and expansion. MCScanX was used to obtain genes in collinear blocks, and JCVI was used to draw images of collinearity. EDTA software was used to identify, annotate, and count complete transposons from the A and B subgenomes of the YZ9102 reference genome and the complete genome of A. hoehnei. Python was employed to statistically analyze genes adjacent to transposons and transposon-associated SVs, followed by GO enrichment analysis of these genes. A resequencing-based phylogenetic tree was constructed using A. hoehnei as the reference genome: BWA and GATK were used to align the resequencing data and generate a SNP-containing VCF file, which was converted to PHYLIP format prior to tree construction with IQ-TREE.

A maximum likelihood (ML) phylogenetic tree was constructed based on the aligned ITS sequences45 using RAxML version 8. BLASTN was employed to align 28,586 probe sequences8 against the Zw61 genome to generate Fasta seq sequences of Zw61. Another phylogenetic tree was constructed using the neighbor-joining (NJ) method, based on the aligned SNP Fasta seq sequences of Zw61 and 588 Arachis species/accessions.

Chloroplast genome assembly and evolution analysis
Total genomic DNA from the 17 species/accessions was extracted using a Plant Genomic DNA Kit (Tiangen Biotech, Beijing) and sequenced on the Illumina HiSeq X ten platform (20× depth). Chloroplast genomes were de novo assembled using the GetOrganelle toolkit89. For Arachis phylogenetic reconstruction and maternal progenitor identification, complete plastomes of the 17 species and 4 outgroups (A. thaliana, C. arietinum, M. truncatula, and G. max) were aligned with MAFFT90, and ML phylogenetic trees were constructed using FigTree91 with the 21 sequences. Jellyfish software was used to analyze the K-mer frequency distribution for heterozygosity, estimate genome size and calculate SNP proportions from Arachis genome sequencing data.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Overall comments\nThere is a lot in this manuscript, the chromosome images are impressive!\nWhilst there is a considerable amount of merit, there are issues that need to be resolved.\nThe use of the term mini-chromosome is not correct. The Arachis chromosome in question is about half the size of most of\nthe other Arachis chromosomes, and, therefore, doesn’t fit in the definition of mini-chromosome - contrast, for instance, the\ncase of chicken mini-chromosomes that are about 10x smaller than chicken macro-chromosomes. The correct term, in the\ncase of Arachis, is the \"A chromosome\", the authors can cite Husted 1933 (Cytological Studies on the Peanut, Arachis. I.\nChromosome number and morphology. Cytologia, 5(1), pp.109-117.) for the original observation of the small chromosome,\nand Bertioli et al 2016 (The genome sequences of Arachis duranensis and Arachis ipaensis, the diploid ancestors of\ncultivated peanut. Nature genetics. 48:438-46.) for the description of how reciprocal translocations between ancestral\nchromosomes 07 and 08 icreated this derived state.\nI was unable to access the full genome sequence reported in the paper (of what the authors refer to as A. stenophylla see\nmore below). Also, I apologize if I have missed anything, but where are the sequences of the oligonucleotide probes DP-1,\nDP-2, etc? Access to the genome and probe sequences is crucial for publication, and to giving a conclusive review\nThe authors should use the original collection numbers of accessions used, e.g., V14167, not newly made-up numbers, e.g.,\nZw55, that obscure the traceability of the germplasm used, make it very difficult to accumulate systematic knowledge on\ngermplasm, and don’t give due credit to those who originally collected the germplasm in the field.\nThe largest issue is a critical issue of species identification. What the authors refer to as Arachis stenophylla, doesn’t appear\nto be that species. There are a number of very clear indications:\n1) The leaf presented as belonging to A. stenophylla (Fig. 6) is ovobate, whereas the leaves of A. stenophylla are linear-\nlanceolate; see the attached screenshot from the monograph and images from herbarium specimens.\n2) The position of what the authors refer to as A. stenophylla in the phylogeny in Figure 7 is unexpected for a species from a\ndifferent botanical section (Erectoides). The position on the phylogeny is consistent with their plants being an A genome\nspecies from the botanical section Arachis\n3) The behavoir of the hybrids described in the paper is consistent with an A genome species. A species from the section\nErectoides would be unlikely to hybridize at all.\n4) In the Genome Sequence Archive (https://ngdc.cncb.ac.cn/gsa/browse/insdc/SRA1714983/SRX21820157) \"WGRS\nArachis stenophylla species”, the link to the Run Accession “SRR26105744” goes through to a project entitled\n\"Resequencing of Arachis villosa species” (https://ngdc.cncb.ac.cn/bioproject/browse/insdc/PRJNA1002116). Arachis\nvillosa is an A genome species of the botanical section Arachis.\nI conclude with a high degree of certainty that what is referred to as A. stenophylla is an A genome species from the\nbotanical section Arachis.", "reviewer_2": "Key results\nThe MS describes generating a complete genome reference for Arachis stenophylla, a wild species of cultivated peanut, and\ncytological evidence to support a hypothesis for the evolution of the Arachis genus. The cytological evidence suggests that\nthe E genome (A stenophylla) is a bridge between ancestral state of chromosome 8 in the A lineage and the mini\nchromosome that\nValidity\nThe interpretation of the cytological data is valid and is corroborated with previous literature. Genome assembly and\ndescription was correct.\nSignificance\nThe main focus of the MS is generating the T2T genome of an E genome Arachis wild species. While the genome is\ncertainly well constructed, the biology of the genome itself is not novel or complex. Assembly and scaffolding of the\nassembly was done with common methodology. The genome itself did not provide additional biological insights, only\nvalidated that the E genome was in between the H,B, K and A species. The MS did not provide evidence for a novel\nmechanism for this evolution.\nData and methodology\nData generation and genome assembly is appropriate. It is beyond this reviewer’s expertise to judge cytological\nmethodology.\nSuggested improvements\nThere are multiple cultivated genome assemblies publicly available in peanutbase.org and in other databases. Those\ninclude multiple wild species, mostly A genome species. There is very little comparative genomics analysis of the E genome\nvs other Arachis genomes. It would be of interest to understand the potential value of this particular genome sequence as it\nrelates to the others outside of the evolution of the genus, which is not of significant interest currently.\nThere are claims that A stenophylla is valuable as a source of novel alleles for cultivated peanut, but there is not data\npresented to showcase this. There is the discussion of novel gene families found, and it would be interesting to understand\nthe expression of those genes as related to other gene networks.\nClarity and context\nThere is a long presentation of cytological interpretation that should be summarized differently than in text.", "reviewer_3": "Peanut is an important oil and food crop. There are many wild species of peanut that can cross directly or indirectly with\ncultivated peanut, which contain many elite resistance genes. In the paper, a set of FISH fluorescent probes were developed\nto analyze the karyotypes of several peanut species, showing the diversity of karyotypes between wild peanut and cultivated\npeanut. Then, using genome sequencing, A. stenophylla was assembled into a gap-free T2T genome. Bioinformatics\ncombined with cell karyotype analysis was used to systematically analyze and predict the evolutionary relationship of\nArachis. The work provided an important theoretical basis for broadening the cultivated peanut gene pool and improving\npeanut varieties through chromosome engineering. I think that this paper can be published after minor revision.\n1. Line 21, Abstract: Arachis species show multiple resistance to pests, diseases and abiotic stresses. “Arachis” should be\n“Wild Arachis”.\n2. Line 66, Arachis comprises 83 species. Here, a reference is necessary.\n3. Line 1260127, “Adu01 had strong green signals in both the centromeric and terminal regions of the short arm”,\nthe corresponding fluorescent probe type should be indicated, and the corresponding picture of this paragraph should be cited.\n4. Line 270, what the difference between A. stenophylla and ZW61 used for sequencing. According to Table S17, the\nheterozygosity rate of ZW61 is 0.88%. The data for “0.37%” should be cited.\n5. Line 352, please confirm the copy numbers. According to Table S12, there are 1581 copies for chr02, and 512 copies for\nchr.06.\n6. Line 367, Table S13 should be cited.\n7. Line 433 and Line 434, the genome size is G or Gb?\n8. Line 686, MS+0.8 mg L -1 NAA+3% sucrose.\n9. Line 717, Du et al. (2018) 30.\n10. Line 750, per MB, or Mb?\n11. Line 827, T=K/(2 × r), where the molecular clock r was 7 *10-9. Please use a uniform format for “x” or “*”.\n12. Line 892, across genomes A, B, F, K and E genomes. How about H genome?\n13. Line 893, why chr.05 was not mentioned?\n14. Figure S25 C, the bar is cM or uM?"}, "author_response": "Thanks for your kind review and critical comments.\nResponse: This is a very critical suggestion, thank you very much. Based on your suggestion, the\ntwo references “Husted 1933 (Cytological Studies on the Peanut, Arachis. I. Chromosome number\nand morphology. Cytologia, 5(1), pp.109-117.) and “Bertioli et al 2016 (The genome sequences of\nArachis duranensis and Arachis ipaensis, the diploid ancestors of cultivated peanut. Nature\ngenetics. 48:438-46.)” were cited, and the term “mini-chromosome” in our manuscript was\nchanged to \"A chromosome\".\nResponse: Thanks for your comments. We re-uploaded the genome assembly and gene annotations\nof Arachis stenophylla (PI 468178) to Figshare\n(https://doi.org/10.6084/m9.figshare.29145581.v3), all the oligo probe sequences including DP-1\nand DP-2 were included in supplementary Table 2. We hope you can access these data this time,\nany question please contact us.\nResponse: Thanks for your comments. We included the original accession number of all wild\nspecies in Table 1, all the information of these species could be traced in the website\n（https://npgsweb.ars-grin.gov/gringlobal/search）based on the PI number.\nResponse. This is a very critical comment. The species utilized in our study were previously\nintroduced from the USA. To preserve the wild species, a dedicated nursery was established,\nwhere each species was planted in a permanent and separate pool with 200 cm in length, 150 cm\nin width, and 50 cm in height. The distance between adjacent pools was maintained at 50 cm\n(Fig.1). In response to your comments, we examined the leaves of A. stenophylla used in the\nmanuscript. The leaf depicted in the manuscript was taken from the middle section of a lateral\nbranch of A. stenophylla plant, as indicated by the yellow arrow in Figure 1a. However, it should\nbe noted that the main stem leaves of A. stenophylla observed in this study were linear-lanceolate.\nWe further compared the phenotypic images of leaves from two accessions of A. stenophylla you\nprovided and found that \"1_reviewer_attachment_2_1738781571\" was similar to those growing\nin our wild species pools (Figures 1a and 1b). Nevertheless, it is important to highlight that the\nleaf length/width ratio for A. stenophylla observed here is significantly less than the reported\nrange of 7-12:1; specifically, our measurements indicated a leaf length/width ratio on its main\nstem ranging from 3-4:1. We can’t ensure that whether these variations were induced by\nenvironments. Furthermore, we also compared the pod phenotypes of A. stenophylla in\nKrapovickas et al. (2007) with those in this study, and they were very similar (Figures 1c and 1d).\nWe added the plant phenotypic image of A. stenophylla in Figure 4 in the manuscript.\nKrapovickas, A., Gregory, W. C., Williams, D. E., and Simpson, C. E. (2007).Taxonomy of the\ngenus Aeachis (Leguminosae). Bonplandia 16.\nResponse: Thank you for your comments and suggestions. From the analysis of the nuclear\ngenome, chloroplast genome and GISH, it is evident that A. stenophylla exhibits a closer\nrelationship to the A genome. But consensus karyotypes and collinear comparison showed A.\nstenophylla is quite distinct from both the A and B genome species. The average percentages of\nsynteny blocks across A, B, and Est genomes were ordered as followed: B-B (84.01%) > A-A\n(67.11%) > Est-A (23.56%) > A-B (18.08%) > Est-B (17.45%). In comparison with the three\nreference T2T genomes Est, YZ9102-A and YZ9102-B, the structural variation number (>=50 bp)\namong the different genomes was ordered as Est- A (47,590.71) > A- B (30,955.2) > Est- B\n(28,716.8) > B-B (23,979) > A-A (22,836). The number of SNP and indels (<50 bp) were ordered\nas Est-A (4,317,609) > A-A (3,682,424) > Est-B (2,122,313) > A-B (1,994,344) > B-B (1,069,269).\nIn comparison with A genomes, similar inversions exceeding 5 Mb occurred on chromosomes 1, 3,\n5, 8, 9 and 10 in Est while for all B genomes, similar inversions found in chromosomes 1, 5, 6, 7\n,\nand 9 of Est. Large segmental translocations or complex variations were also particularly\nobserved on chromosomes 7 and 8. Based on the SCVs of chromosomes 7 and 8, the Est of A.\nstenophylla seems bridging the B and A genomes. Please see Results section, line 391-516.\nAs we do not possess germplasm samples of A. stenophylla corresponding to those depicted in\nyour provided image nor any other germplasm of A. stenophylla, so we can't confirm it further. To\naccurately reflect the genomic attribution of A. stenophylla, we have revised its genome symbol\nwithin Fig. 7 and throughout the manuscript by using \"Est \" to specifically denote this genome\ndistinguishing it from other E genomes. Of course, We also hope that reviewers will offer\nadditional suggestions regarding genomic attribution studies and evolutionary research related to\nA. stenophylla based on both the genomic data and phenotypic images we provided. We will\nfurther improve it according to your suggestions.\nResponse: Thanks for your critical comment. Our study did reveal that A. stenophylla is more\nclosely related to the A genome species, and the hybridization between cultivated peanut and A.\nstenophylla was easy. But this species also showed unique variations, TE contents, gene families\nand synteny level in comparison with the A genomes. The most typical distinction is that A.\nstenophylla did not have the “A chromosome” as present in all other A genome species, thus the\ngenome size and chromosome structure of 8Est was quite different from 8A. We are also very\nconfusing about the classification of this genome, wish your more critical comments on solving\nthis problem.\nResponse: Thank you for your comments and suggestions. We used our T2T genome as reference,\nboth SNPs and INDELs were called for both A. stenophylla \"SRX21820157\" and A. villosa\n\"SRX21763288\" as you suggested. The results indicated that the two genomes exhibit large\namounts of SNPs and InDels (Figure 2a). Many low coverage regions of \"SRX21820157\"\nsequences on the T2T genome of A. stenophylla were observed suggesting distinct sequence\nconstitutions in the two genomes (Figure 2b).\nAs mentioned above, our comparative genomic analysis demonstrated that the A. stenophylla\ngenome shows a closer relationship with genome A. But regarding genome size, A. stenophylla is\n1.61Gb, while the A subgenome of YZ9102 is 1.16Gb; other species such as A. duranensis, A.\nipaensis, A. cardensii, and A. stenosperma were 1.08Gb, 1.35Gb, 1.24Gb, and 1.33Gb,\nrespectively (Supplementary Table 20). Thus A. stenophylla possesses the largest genome among\nthe current diploid wild species. Regarding karyotype, the arm ratio of chromosome 8 in A.\nstenophylla is 4.77, with a length of 115.65 Mb and classified as subtelocentric, which\nsignificantly contrast with the metacentric chromosome \"A chromosome\" 8A in A genome. A.\nstenophylla has two pairs of chromosomes with both 45S and 5S rDNA loci simultaneously\n(Figure 3), and no other species exhibiting this characteristic within section Arachis as reported.\nRegarding synteny level, the length percentage of synteny blocks between Est-A was quite lower\nthan A-A. Based on the above findings, we proposed that A. stenophylla did not belong to any\ngenome categorized as A, B, D, F, K, and G currently, but whether this genome belonged to E, we\nare not sure, so we modify its genome to Est in order to distinguish it from both A and E. We\nextended comparative genomic analysis results among more reported genomes in the text. Please\nsee the Results and Discussion sections.\nResponse: Thank you very much for your kind review and suggestions. As you suggested, in order\nto improve our manuscript, we made a thorough revision focusing on highlighting the four\ninnovations. First, we established a consensus karyotype for Arachis species, which enabled all\nthe pseudomolecules corresponded to the actual chromosomes of A. duranensis and determined\nthe Hgs of the A, B, F, K, E and H genomes at the first time. Based on this consensus karyotype,\nwe identified many SCVs during genome evolution of Arachis, and found that the Est of A.\nstenophylla seems to bridge the B and A genomes. Second, we assembled a T2T genome for the\ndiploid wild species with the largest genome among Arachis diploid species, which was not only\nthe first T2T genome for a wild peanut, but also represent the third genome assembled in Arachis\nspecies apart from A and B genomes. Thirdly, our research has revealed the origin of \"A\nchromosome\", and identified SVs, TE variations and outcrossing as key factors driving genomic\nevolution in Arachis species. We proposed a model for genomic evolution based on these findings.\nLastly, previous studies have indicated that A stenophylla harbors numerous valuable\ninsect-resistant genes against pests such as Armyworm, Corn Earworm, and Groundnut aphid\n(Sharma et al. 2003; ICRISAT, 1990). Our investigations revealed that A. stenophylla exhibits\nhigh resistance to web blotch disease; furthermore, we identified four candidate resistance genes\nassociated with this trait, and created an artificial hexaploid peanut by crossing cultivated peanut\nwith A. stenophylla for peanut breeding. According to your suggestions, we have conducted\nexperimental supplementation and analytical enhancements while summarizing and refining our\nmanuscript accordingly. Please see the Results section and the Discussion section.\nResponse: Thanks for your valuable suggestions. According to your suggestion, we downloaded a\ntotal of eight genomes from peanutbase.org and other databases. These include A. ipaensis, A.\nduranensis, A. cardensii, A. stenosperma, Tifrunner (A. hypogaea), YZ9102 (A. hypogaea), STQ\n(A. hypogaea) and A. monticola. Through comparative genomic analyses involving these genomes,\nwe provided a more detailed explanation of the variation processes occurring in chromosomes 8B,\n8Est, and 8A. Furthermore, we identified SVs of Est compared to A or B genomes, and discovered\nspecific genes of Est genome. Notably, gene mutations resulting from SVs or SNPs were also\nrevealed. These genes enriched in pathways related to glutathione metabolism, ascorbate and\naldarate metabolism etc. Glutathione and ascorbate are well-known antioxidants in plants and\nare believed to be linked to plant stress resistance and adaptability. These findings likely\ncorrespond with the resistance mechanisms of A. stenophylla against sticky disease, corn earworm,\nand peanut aphids. In addition, it was also found that SVs, TE variations and outcrossing played\nan important role in driving the evolution of the genus Arachis. Please see line 391-544 in Results\nsection and line 619-718 in Discussion section.\nResponse: Thank you for your critical comments and suggestions. We identified 367 specific gene\nfamilies in A. stenophylla. Through gene enrichment analyses, we found that certain genes may be\nassociated with the disease resistance and insect resistance of A. stenophylla. By identifying web\nblotch and conducting transcriptome analysis, we identified 46 candidate genes involving SVs\nassociated with web blotch resistance; among these, three genes are likely to contribute to the\nmechanisms of web blotch disease resistance in peanuts. Additionally, a pathogenesis-related\nprotein (PR10) gene Ast01G035260.1 was discovered as potentially playing a significant role in\nconferring resistance to web blotch. To utilize the advantageous genes of A stenophylla for peanut\nbreeding, we developed an artificial hexaploid peanut by crossing cultivated peanut with A\nstenophylla.\nResponse: Thanks for your suggestions. We have summarized and revised the cytological\ninterpretation.\nResponse: Thanks for your insightful review and valuable suggestions concerning our manuscript.\nIn response to your suggestions, we have revised the manuscript in detail.\nResponse: Thanks for your suggestions. We revised it to “Wild Arachis species”, Please see line\n21.\nResponse: Thank you very much. “Arachis comprises 83 species” was cited from Leal-Bertioli et\nal. (2024), reference 8. We added references, please see line 66.\nResponse: Thanks for your suggestions. These signals were from the oligonucleotide signals\n(SSON), we revised it and cited the corresponding picture of this paragraph. See line 147-148.\nResponse: Thanks for your suggestions. ZW61 refers to A. stenophylla and is derived from the\nsame material as this species. In the study, we utilized two distinct plants: one for T2T sequencing\nassembly and another for resequencing at a depth of 30×. The heterozygosity between different\nplants may vary; furthermore, their differing sequencing depths could lead to discrepancies in\nestimated heterozygosity levels. To avoid confusion, we reported a heterozygosity rate of 0.37% in\nthe manuscript. According to your suggestions, we have revised. Specifically, Table S17 has been\nrenumbered as Supplementary Table 24 following the order of tables presented in the manuscript.\nPlease see Supplementary Table 24.\nResponse: We are very sorry for the mistakes in labeling the copy numbers of 5S rDNA on chr02\nand chr06. The copy numbers on chr02 are 1581 copies, and the copy numbers on chr.06 are 512\ncopies. Thank you for pointing out the mistakes. The Table S12 (Supplementary Table 16) has\nbeen modified, please see line 372.\nResponse: Thank you very much. We revised and cited the table, and Table S13 was revised to\nSupplementary Table 17, please see line 456.\nResponse: Thanks for your suggestion. They should be “Gb”. We revised them. Please see line\n529.\nResponse: Thanks for your suggestion. We revised it. Please see line 794.\nResponse: Thank you. We revised it.\nResponse: Thank you. They should be “Mb”, we revised them in the manuscript.\nResponse: Thank you. According to your suggestion, we revised it.\nResponse: Thank you for pointing out the problem, we added the H genome, please see line 1240.\nResponse: Thank you, chr.05 should also be mentioned, we revised it. Please see line 1240.\nResponse: Thank you point out our mistakes. It should be μm, the bar has been revised. Please see\nSupplementary Fig. 33C."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you for the authors for responding to my comments.\nThe authors have obviously put a lot of work into this research. The issue that must be resolved is the identity of the plant\nthat the authors thought was A. stenophylla. The aim of my comments is to try to help the authors determine the real identity\nof this plant—a totally feasible aim. Then they can reinterpret their data and observations and have a good publication.\nWith permission of the editor, I shared images of the plant with three experts. These are summaries of their comments:\n\"This plant is not A. stenophylla which has lanceolate leaves, whereas this plant has much more ovate leaves.\"\n“the plant shown in the upper picture is certainly not A. stenophylla, which has linear-lanceolate leaflets (lenght/width\nrelation 7-12/1, in the 1994 monograph key).\"\n\"Arachis stenophylla is listed among the species of section Erectoides tightly associated to A. paraguariensis. It was\nsuccessfully crossed by Gregory & Gregory to A. martii (0.07% pollen viability) and to A. paraguariensis (16.8%) only.”\n\"A quick mitotic chromosome preparation showing the A-pair will exclude A. stenophylla.\"\nThis blog post with photos of a specimen that was confirmed to be A. stenophylla was brought to my attention. It is very\ndifferent to the one in the photo provided by the authors\nhttps://rubens-plantasdobrasil.blogspot.com/2015/05/fabaceae-arachis-stenophylla-krapov-wc.html\n\"the flowers appear to be yellow. If this is the true flower color, then it is not stenophylla\"\n\"Although I have never attempted hypogaea X stenophylla, I think it is doubtful, but I have made enough wide crosses that I\nwill not say impossible. If I was reviewing the paper I would as for more proof of the plant ID.\"\nI don’t think this plant is A. stenophylla. I think it is an A genome species in Section Arachis. The chromosomes have almost\nall the rearrangements that are characteristic of an A genome species, and are derived states compared to the B genome\nspecies (the fact that they are derived states can be deduced by the distribution of repetitive DNA, this is explained in Bertioli\net al 2016). In the annexed file there are a set of dot-plots comparing homologous chromosomes of “A. stenophylla” (vertical\naxis) with A. duranensis (horizontal axis) that support these conclusions:\nChr 1 shares the inversion of both arms with A. duranensis\nChr 2 is co-linear with A. duranensis chr 2, both retaining the ancestral state\nChr 3 is co-linear with A. duranensis chr 3, both retaining the ancestral state\nChr 4 is co-linear with A. duranensis chr 4, both retaining the ancestral state\nChr 5 shares the large inversion in the lower arm of A. duranensis chr 5\nChr 6 shares the large inversion in the upper arm of A. duranensis chr 6\nChromosome 7 shares the derived state with A. duranensis, the product of complex reciprocal translocations between\nancestral 7 & 8.\nChromosome 8—The gene-rich A chromosome—is present in the A. stenophylla. A08 is derived from the same reciprocal\ntranslocation between the ancestral 7 & 8 that gave rise to A07. BUT, the upper arm of the “stenophylla” A08 is expanded\ncompared to A. duranensis, and it has two inversions relative to A. duranensis (At least one of them being real—which is\nindicated by the curve in the dot plot)\nThe ”A. stenophylla” A09 retains the ancestral state, lacking the inversion in the lower arm of A. duranensis. This is real, not an artifact of assembly (this is apparent in the curve of the inversion)\nChr 10 is co-linear with A. duranensis chr 10, both retaining the ancestral state\nThe “A. stenophylla” isn’t A. stenophylla. It appears to be an A genome Arachis, perhaps a basally diverging one. There are\ntwo ways of finding out using data that you already have:\n1) Extract the SNPs in the “A. stenophylla” genome for Affy probes in the Excel file\n“AllDiploids_Affy_UPAC_fasta_V02.xlsx”. The marker probe sequence information is in the file\n“Axiom_Arachis2_AnnotationFile.r1.csv”. Only a subset of high quality extracted SNPs will be necessary. Then create a\npseudo sequence of the “A. stenophylla” SNPs together with all the ex-situ samples of the section Arachis and make a tree.\nThe dataset provided is from this paper: https://bsapubs.onlinelibrary.wiley.com/doi/pdfdirect/10.1002/ajb2.16357\nThis new tree with the “A. stenophylla” is very likely to identify the true identity of the plant to the accession, or very close!\n2) Extract the ETS, psbA-trnH, rpoB-trnC, ycf1, and ITS sequences from the “A. stenophylla” and build a tree with\nrepresentatives from all the species in the genus Arachis from the data made available with this paper:\nhttps://academic.oup.com/aob/advance-article/doi/10.1093/aob/mcaf139/8209677#528667952\nThis will identify the species, which will be helpful if the “A. stenophylla” is outside the section Arachis.\nHoping this helps. Once a firm answer as to the identity of this plant is found, your paper can be altered accordingly and be a\nvaluable contribution!\nDavid Bertioli\nThe following is a statement of my sincere philosophical beliefs, in which I conflict with the Nature Portfolio’s stated policies.\nThis statement is integral to this review and is included as part of the commitment to transparency of open review. It does not\nrequest or require a response or comment from the authors of the paper under review.\nIn keeping with the Socratic tradition and Merton’s norms, I have evaluated this manuscript solely on its scientific merit. I\nbelieve that knowledge advances through open inquiry, fallibility, and a commitment to truth, not through adherence to\nprescribed social or political frameworks. Fairness, as expressed in Merton’s norm of universalism, is essential in science.\nNature Portfolio’s policies on ‘Diversity,’ ‘Inclusion,’ and ‘Gender’ embed a narrow, culturally specific, and politicized\nworldview. This framework conflicts with classical scientific norms—including all of Merton’s norms—and has demonstrably\nhindered the pursuit of objective understanding while contributing to the erosion of public trust in science. I have therefore\nnot allowed these policies to influence my review—for example, I convened a ‘manel’ of experts, chosen solely for their\nexpertise in the genus Arachis.", "reviewer_2": "The authors have made significant improvements to the MS based on the reviewers comments and criticisms. The edited\nMS is significantly improved and is ready for publication", "reviewer_3": "1. Fig S19 \"FISH using CSCOLs L6A-1 and L6A-1 in PI 497572.\" should be L6A-1 and L10A-1.\n2. The format of the \"Subtelomere position (bp)\" column in Supplementary Table 11 is inconsistent."}, "author_response": "Response:Dear Professor David Bertioli, thank you very much your critical comments and\nvaluable analytical results, literatures as well as the relative data information. Based on your\nsuggestions, we reanalyzed the genome identity of the accession PI 468178 using both the ITS\nsequences and target SNPs and clarified its genome identify.\nFirst, based on ITS sequences of Arachis L. reported by Inglis et al. (2025), we extracted the ITS\nsequence of PI 468178 using MAFFT version 7 and constructed a phylogenetic tree for the\nnucleotide dataset via RAxML version 8. The results showed that PI 468178 (\"A. stenophylla\")\nand A. hoehnei formed a single clade (Supplementary Fig. 35), which is positioned at the base of a\nlarger clade encompassing all A genome species.\nSecond, utilizing the 28,586 probe sequences provided by you, we aligned them with the\ntelomere-to-telomere (T2T) reference genome of PI 468178 (\"A. stenophylla\") via BLASTN to\nextract the target SNPs and generated consecutive SNP Fastaseq sequences. Then we used\nMAFFT software align the Fastaseq sequence of PI 468178 (\"A. stenophylla\") with the 588\nFastaseq sequences of Arachis species you provided. The Neighbor-Joining (NJ) method was then\napplied to construct a phylogenetic tree (Supplementary Fig. 36). Consistent with the first\napproach, PI 468178 clustered with multiple A. hoehnei accessions, showing the closest\nrelationship with accession 9094 Valls B 776 M18. Combined with previous cytological data\nshowing consistent rDNA site distribution between Zw61 and A. hoehnei (PI468150) (Du et al.\n2016), we conclude that Zw61 should be an accessions of A. hoehnei in section Arachis, rather\nthan A. stenopylla (PI 468178), this material might have gone errors during the germplasm\nintroduction or propagation.\nPreviously, A. hoehnei genome is unknown, but our study reveals unique features. Firstly, it is\ncurrently the largest known genome among diploid wild Arachis species. Secondly, chromosome\n8A is not small enough. Thirdly, it contains chromosome 7/8 translocations and lacks inversions.\nAlthough chloroplast and nuclear genomic SNP analyses indicate that it is closely to A, yet\nexhibits significant differences, including SV, SNP, etc., indicating ancient divergence and\ndistinctiveness from A. Based on these results and your suggestion, we designated A. hoehnei\ngenome as “A'”, which represents a basally diverging form of A-genome worthy of further\ninvestigation.\nResponse:Thank you very much for your comments.\nResponse:Thank you. We have made corrections.\nResponse:Thank you. We have revised."}, {"round": 3, "reviewer_comments": {"reviewer_1": "Congratulations to the authors for their scholarship in working out the true identity of their wild peanut accession, which\nturned out to be Arachis hoehnei (not A. stenophylla as they had originally thought). With the identification of the plant's true\nidentity, the authors have adjusted the conclusions of their extensive study and made very valuable contributions. This study\nwould fit well in Nature Genetics, and is overdimensioned for Nature Communications."}, "author_response": "Response: Thank you very much. We are sincerely grateful for your generous recognition and\nconstructive comments on our study."}]

Decision letter