A near complete genome of <i>Arachis monticola</i>, an allotetraploid wild peanut

Abstract

Peanut (Arachis hypogaea L.) is the most economically important oilseed legume crops throughout the world. Most Arachis species are diploid, but two are heterotetraploid: one cultivated species (A. hypogaea) and one wild relative (A. monticola). A. monticola was an important intermediate between diploid wild peanut species and tetraploid cultivated peanut varieties (Zhuang et al., 2019). We released the first A. monticola genome in 2018 (Yin et al., 2018), but the quality was not high enough. The development of long-read sequencing technologies has enabled the assembly of higher-quality genomes, even at the telomere-to-telomere scale, such as Arabidopsis thaliana (Wang et al., 2022) and Oryza sativa (Song et al., 2021). Here we present Amon2.0, an updated high-quality genome assembly of A. monticola generated by combining Nanopore ultra-long, Hi-C reads and MGI short. The heterozygosity was estimated at 0.8%. The genome was de novo assembled, and then polished with both long and short reads. After scaffolding with Hi-C reads, the assembly has 46 scaffolds, with a total size of 2.56 Gbps and an N50 value of 137.6 Mbps, and 99.6% of the sequences were assembled into 20 chromosomes (Table S1; Figure 1b). The final genome assembly contained only 34 gaps, which was much fewer than the previously published tetraploid peanut genome (Bertioli et al., 2019). In total, 11 chromosomes have both complete telomeric sequences and the remaining 9 chromosomes have one complete telomeric sequence. All chromosomes have centromeric sequences, including 13 chromosomes with complete centromeric sequences. A total of 2 147 185 085 bps (83.96%) was annotated as repeat elements, which were mainly long terminal repeat (LTR) retroelements (65.3%) and DNA transposons (12.03%). The highest proportion of LTR retroelements were Gypsy sequences (67.1%). LTR Assembly Index (LAI) assessment demonstrated that Amon2.0 achieved gold-standard quality for repeat regions with an LAI score of 21.27. Totally, 75 226 protein-coding genes from 35 556 gene families were predicted. Specifically, 34 728 and 40 282 genes were identified in sub-genomes A and B, respectively. Of these genes, 74.6% were functionally annotated and 91.2% contained at least one known structural domain. The complete and duplicated Benchmarking Universal Single-Copy Orthologs scores were 99.3% and 91.5%, respectively, corresponding to 96.2% and 96.7% in sub-genomes A and B, respectively. The densities of genes, DNA transposons and LTR/Copia elements were higher near the telomeres, whereas LTR/Gypsy elements were mainly distributed near the centromeres (Figure 1a). Compared with the previous A. monticola genome release (Amon1.0), Amon2.0 filled 248.1 Mbps of unknown sequences, with 243.6 Mbps and 180.8 Mbps of sequences anchored in sub-genomes A and B, respectively (Table S2). Amon2.0 was highly continuous, with a contig N50 value of 58.3 Mbps. Amon2.0 had an assembly consensus quality value score of 34.2, corresponding to >99.9% accuracy in consensus base calls. All of these measures indicated that the contiguity and completeness of both sub-genomes in Amon2.0 were superior to those of Amon1.0. Collinearity analysis of Amon2.0 and Amon1.0 indicated the similarity was high between the corresponding chromosomes (Figure 1c; Figure S1). There was a lower similarity between homologous chromosomes. We identified an inconsistent region in chromosome A03 (from 14.94–32 Mbps in Amon2.0) (Figure 1d). This region was verified with the alignment of long reads. (Figure 1e,f). This indicated that Amon2.0 eliminated some assembly errors of Amon1.0. Compared with Amon2.0, 10 818 structural variations (SVs) were detected in the cultivated peanut Tifrunner (Table S3). Some SVs influence the structure and expression of nearby genes (e.g. LRK10L and ARF3) (Figure S2). We next performed RNA sequencing in five A. monticola tissues. In five tissues, 57.5%–64.8% of all genes in the genome were expressed (FPKM>0), corresponding to 61.3%–68.6% and 54.7%–61.8% genes in sub-genomes A and B, respectively (Figure 1g). In total, 71.7% of all predicted genes were expressed in at least one tissue, corresponding to 75.6% and 68.6% of the genes in sub-genomes A and B, respectively. Based on gene family data, all sub-genomic genes were further divided into four groups: A-specific (genes only present in sub-genome A), A homologue (sub-genome A genes present in both sub-genomes), B-specific (present only in sub-genome B) and B homologue (sub-genome B genes present in both sub-genomes); there were 7682, 25 237, 10 718 and 27 162 genes in these groups, respectively. Overall, A homologue and B homologue genes were expressed at significantly higher levels than A-specific or B-specific genes (P < 0.05, Wilcoxon rank sum test) (Figure 1h). We further investigated the expression levels of 16 007 single-copy orthologous genes. Preliminary results showed a trend of tissue-specific asymmetric gene expression for paired genes belong to two sub-genomes (Figure 1i). For example, the orthologous gene LECC1 (SL-I) was higher expressed from sub-genome A in the fruits (FPKMAM09G34310 = 9657, FPKMAM19G32920 = 3833, Figure 1j). SL1 is related to a gene encoding a reportedly drought-inducible alpha-methyl-mannoside-specific lectin. In conclusion, this study introduces Amon2.0, a near-complete, highly accurate genome assembly for A. monticola. Comparison of this genome assembly with the previous A. monticola reference genome clearly demonstrated the increased continuity, completeness and accuracy of Amon2.0. The unprecedented quality of this genome enabled us to observe tissue-specific asymmetric gene expression patterns between the A. monticola sub-genomes. This genome assembly will serve as a fundamental basis for further understanding of the domestication and evolutionary histories of Arachis spp. and the family Fabaceae more broadly. Furthermore, this genetic resource will contribute to functional genomics and future molecular-assisted breeding in these economically-important legume crops. The authors declare that they have no competing interests. D.M.Y. designed this experiment. H.Z.X., K.Z., S.Y.H., A.C., K.K.Z., L.Z., D.Q., R.R., F.P.G., Z.F.L., X.L.M. and X.G.Z. conducted work. H.Z.X., D.M.Y., C.C.W., X.Y.Z. and R.K.V. edited and revised the manuscript. This work was supported by grants from NSFC-Henan United Fund (No. U22A20475), Key projects of Henan Province (No. 222301420026; 221111110500; HARS-22-05-G1). The genome has been deposited in the National Genomics Data Center (https://ngdc.cncb.ac.cn). The transcriptomic profile is available in the OMIX (OMIX004792). The raw sequence data has been deposited in the Genome Sequence Archive (CRA012330 and CRA012333). Figure S1 Dot plot of synthetic similarity in genome sequences between Amon1.0 and Amon2.0. Figure S2 Structure and expression of SV-related genes LRK10L and ARF3 in Tifrunner. Table S1 Chromosome-wise statistics of genome assembly Amon2.0. Table S2 Comparative assessment Amon2.0 and Amon1.0 assemblies. Table S3 Statistics of SVs between Tifrunner and Amon2.0. 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