Background & Summary

Soybean is an important crop that is responsible for about 50% of the world’s oilseed production (www.fao.org), and a source of high-quality protein for animal feeds. Cultivation of soybean has experienced specific selections for the last 4,000 years, yielding over 45,000 Glycine max (G. max) accessions1. The construction and subsequent targeted improvement of the reference genome assembly for G. max Williams 82 (W82), has greatly enhanced genome contiguity and has in turn promoted research on soybean2,3. However, recent genomic advances in many plant and animal species have shown that a single reference genome is insufficient to capture and represent the variations that exist within the population of the corresponding species4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20. Such is also true in soybeans. Resequencing and de novo assembly of wild and cultivated soybean accessions identified hundreds of genic presence-absence variations (PAVs) or SVs affecting agronomic genes compared to W82 genome7,21,22,23,24,25,26. The recent construction of the soybean pan-genome also discovered over 120 K non-redundant SVs27. Thus, we endeavor to enhance the genomic resources available for furthering soybean functional research and breeding by constructing reference-grade genome assemblies from cultivars found in different latitudes and exhibiting different agronomic traits. In this data descriptor, we report the sequencing, genome assembly, annotation, and genomic variation resources generated for the assembly of 8 reference-grade soybean genomes.

Methods

Sample selection, collection, and nucleic acid extraction

Eight soybean accessions were selected for this project (Table 1). Aside from IGA1003, which is a wild soybean, the remaining seven were cultivated soybeans. These eight soybeans showed different phenotypic traits including flowering color, pubescence color, maturity time, seed shape, and hundred-grain weight (Fig. 1a, Supplementary Table 1). The eight soybeans were typically grown in different latitudes with IGA1007 and IGA1005 typically grown in southern China (around 22°N) and Huanghuai region (around 31°N), China, respectively (Fig. 1b). In terms of traits, we have included a high salinity tolerant accession (IGA1001) and a high yielding accession (IGA1004). We do note that IGA1003, IGA1005, and IGA1008 were sequenced and assembled previously. IGA1003 was sequenced and assembled using only Illumina sequencing data7. IGA100528 and IGA10083 had published genome assemblies but we have included these materials allowing for assessing genomic variations within a particular accession.

Table 1 Selected 8 soybean accessions.
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Fig. 1
figure 1

Phenotypic variation and geographical distribution of the 8 soybeans. (a) 8 soybean plants after growing 35 days showing differences in growth, leaf shape, stem thickness, and seed color and size; (b) Geographical distribution of the 8 soybeans.

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Seeds from each accession were grown in the greenhouse at Northeast Institute of Geography and Agroecology. Five seeds were planted with 14 hours of light and temperature controlled at 28 °C during the day time and 20 °C during the night time. Soil composition was 8:5:3 ratio of peat:vermiculite:perlite mixed with 5 g of phosphate. Plants were grown until the V2 growth phase and the leaves were harvested from the top.

Genomic DNA was extracted from 1 g of leaf tissue harvested from V2 growth period using CTAB method. Quality of the genomic DNA was assessed using the Qubit Fluorometric system (ThermoFisher) and gel electrophoresis system. Total RNA was isolated from root, stem and leaf separately using TRIzol. Quality of the RNA was assessed using Qubit Fluorometric system and Agilent 2100 Bioanalyzer (Agilent). A quality of RIN >7 was considered high quality RNA.

DNA sequencing library construction and sequencing

DNA fragments >20 kb was selected for PacBio library preparation using BluePippin (SAGE). PacBio library was prepared using SMRTbell Template Prep Kit-SPv3 following manufacturer’s recommendations (Pacific Biosciences). The library QC was performed using Qubit and Agilent 2100. The final library was sequenced on the Pacific Biosciences Sequel system. For Illumina sequencing library construction, the genomic DNA was fragmented to 300–500 bp using Covaris M220 (Covaris). Illumina library was prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina following manufacturer’s recommendations (NEB) and sequenced on the Illumina system with PE150 format. We generated 4–6.74 million PacBio reads29,30,31,32,33,34,35,36 with average subread length N50 of 14Kb and 296–368 million Illumina short reads (Table 2)37,38,39,40,41,42,43,44. The estimated sequencing depth based on soybean genome size was 50X Illumina short reads and 50X of PacBio long reads.

Table 2 Sequencing output of each library type for the 8 soybean accessions.
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RNA sequencing library construction and sequencing

RNA from each tissue was pooled in equal molar to produce a mixed RNA sample. mRNA was enriched using Poly(A) mRNA Magnetic Isolation kit (NEB) followed by RNA-seq library construction using NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB) following manufacturer’s recommendations. Sequencing was performed on the Illumina system with PE150 format to generate 10.43–14.61 Gb of data (Table 2)45,46,47,48,49,50,51,52.

Hi-C sample preparation, library construction and sequencing

About 1.5 g of young leaves were used for Hi-C library construction as described in previous reports with some modifications53. Briefly, leaf samples were cross-linked with 3% formaldehyde for 45 minutes in vacuum at 4 °C and stopped using 0.4 M glycine. Leaf pellets were then pulverized in liquid nitrogen followed by resuspension in the nuclei isolation buffer (NIB). The cross-linked nuclei were treated with 0.3% SDS and neutralized with 3% Triton X-100. The resulting DNA was digested with MboI (NEB) overnight at 37 °C and the reaction was stopped with heat inactivation at 65 °C. Restriction fragment ends were fixed with Klenow and labeled with biotinylated cytosine nucleotides using biotin-14-dCTP (TriLINK). Blunt-end ligation was carried out using T4 DNA ligase incubated at 16 °C overnight. After ligation, the cross-linking was reversed by proteinase K (Thermo) overnight at 65 °C. DNA purification was performed using DNeasy Plant Mini Kit (Qiagen) according to manufacturer’s instructions. Purified DNA was sheared to a length of ~400 bp using Covaris M220 (Covaris). Hi-C ligated junctions were captured by Dynabeads MyOne Streptavidin C1 (Thermofisher) according to manufacturer’s instructions. The Hi-C sequencing library was prepared using NEBNext Ultra II DNA library Prep Kit for Illumina (NEB) following manufacturer’s instructions. Fragments between 400 and 600 bp were sequenced on the Illumina platform with PE150 format to generate 46–57 G of data (Table 2)54,55,56,57,58,59,60,61.

De novo genome assembly

De novo assembly was performed with PacBio sequencing data using FALCON62 setting length_cutoff = 3000–13000 and length_cutoff_pr = 3000 and CANU63 setting correctedErrorRate = 0.039–0.04. We further improved the assembly by merging complementing contigs between FALCON and CANU using CANU assembly as the basis64. The final contigs were assembled into chromosomes with Hi-C data using LACHESIS v.c23474f65. The assembly was corrected with PacBio long reads using Arrow in SMRTLink 5.066 and Illumina short reads using Pilon v1.2267. The final genome assemblies ranged between 986.1 Mb and 1001.3 Mb with more than 98.7% of the genome anchored to 20 chromosomes. The contig N50 were between 1.4 Mb–6.1 Mb and the scaffold N50 were between 48.8 Mb–50.7 Mb (Table 3)68,69,70,71,72,73,74,75.

Table 3 Genome assembly, BUSCO evaluation and remapping assessment of the 8 soybean accessions.
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Genome annotation

Repetitive elements were first annotated before other features. Tandem Repeat Finder v.4.0976 was used to annotate tandem repeats. LTR_FINDER77 was used to build a LTR-retrotransponson library and RepeatModeler v.1.0.10 (http://www.repeatmasker.org/RepeatModeler.html) was used to build a de novo repetitive element library. The above libraries and Repbase78 were used by RepeatMasker79 to annotate repetitive elements. About 50% of each genome was annotated with repetitive elements (Table 4), which is similar to published soybean genomes3,22,28. After repetitive sequences were masked, protein-coding gene annotation was performed utilizing ab initio-, homology-, RNA-sequencing-, and Iso-seq-based methods. Augustus v.3.380 and Glimmer v.3.0.481 were used for ab initio gene prediction. For homology-based annotation, protein sequences from Glycine max Williams 82, Glycine soja, Arabidopsis thaliana, Arachis duranensis, and Cajanus cajan were obtained from NCBI and aligned to each of the 8 genomes using TBLASTN82. Exonerate was used to build gene structure based on the Blast results. For RNA-seq based gene prediction, short reads were mapped to their respective genome using TopHat v2.1.183 and the gene structure was predicted using Cufflinks v2.2.184. For Iso-seq based gene prediction, reads were mapped to IGA1008 assembly using GMAP v2016-09-1485 and TransDecoder v4.1.0 was used to filter for high quality gene models. Lastly, a consensus gene set was generated by integrating gene annotations from each source using MAKER86. With this process, we identified 57,286 to 58,392 protein coding genes (Table 4).

Table 4 Genome annotation and BUSCO assessment of the 8 soybean accessions.
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Non-coding RNA (ncRNA) annotation was performed using INFERNAL87 based on Rfam v.14.1 models. tRNA was annotated using tRNAscan-SE v.1.3.188. rRNA annotation was performed using BLASTN with known soybean rRNA sequences.

BUSCO evaluation of genome and annotations

BUSCO (Benchmarking Universal Single-Copy Orthologs) evaluation was performed on the genome assembly and the gene annotations using BUSCO v.3.0.289 with embryophyta_odb9 data set.

Variation detection

Whole genome alignment based variation detection was performed by aligning each genome to IGA1008 using MUMer490 with parameters–mum –l 40 –c 90. The MUMer alignment was filtered using delta-filter with parameters --1. SNVs, small Indels were called using show-snps with parameters -rlTHC. Large SVs were called using custom scripts MumSV (https://github.com/jeff-sc-chu/MumSV) based on output from show-coords with parameters -THrcl. Alignment gaps associated with assembly gaps were filtered away. Presence-absence variations (PAVs) were assessed based on large insertions and deletions (>100 bp) using BLASTN against the reference genome. A deletion or insertion is considered a PAV if BLASTN alignment to the reference genome is <50% query coverage or <90% PID. Accession specific SNP, Indel, and SVs were identified by selecting variants not overlapping any variants of the same class in all other genomes. Using the above process, we identified 1.86–4.27 million SNVs, 0.44–0.92 million small indels, 11.75–25.33 thousand large indels (>100 bp), 706–3006 translocation events, and 200–413 inversion events (Table 5).

Table 5 Genomic variation of 7 soybean accessions compared with IGA1008.
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Data Records

The sequencing data, genome assembly, and genome annotation of the 8 soybeans have been deposited in NCBI under the BioProject PRJNA561626. This includes whole genome sequencing data from the PacBio Sequel platform29,30,31,32,33,34,35,36 and from the Illunina platform37,38,39,40,41,42,43,44; RNA sequencing data from the Illumina platform45,46,47,48,49,50,51,52; Hi-C library sequencing data from the Illumina platform54,55,56,57,58,59,60,61; and genome assembly sequences and annotations68,69,70,71,72,73,74,75. The individual BioProject, BioSample, and SRA accession IDs are also listed in Supplementary Table 2. Gene annotations in GFF3 format, one-to-one gene correspondences between IGA1008 and W82_v4, and the coordinates of SNPs, Indels, and PAVs are available on Figshare with https://doi.org/10.6084/m9.figshare.c.5106161.v491. We have also detailed the resources available on Figshare in Table 6. The plant materials used in this study are available from the authors upon request.

Table 6 Description of file hosted on the Figshare record (https://doi.org/10.6084/m9.figshare.c.5106161.v4) accompanying this paper.
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Technical Validation

Genome assembly quality assessment

The final assemblies of the 8 genomes ranged between 986.1 Mb and 1001.3 Mb with more than 98.7% of the genome anchored to 20 chromosomes (Table 3). The genome assembly size is similar to its respective k-mer estimation (Table 3) and also comparable to soybean genomes, such as W82_v43, ZH1328, and W0526, which were assembled using similar sequencing technologies. The contig N50 were between 1.4 Mb–6.1 Mb and the scaffold N50 were between 48.8 Mb–50.7 Mb (Table 3)68,69,70,71,72,73,74,75. The number of gaps ranged between 830 and 2922 (Table 3). Each unspanned gap in the assembly was arbitrarily set with 500 bp of Ns. As a comparison, the current soybean reference Williams 82 version 4 genome has a genome size of 978 Mb with contig N50 of 0.41 Mb, scaffold N50 of 20.44 Mb, and 8920 gaps3.

We further assessed the quality of our genome assemblies in a number of ways. First, the presence of centrimeric repeats CentGm-1/2 were found in all 20 chromosomes for all genome assemblies. We assessed the completion and accuracy of the assemblies using BUSCO89 and re-mapping of Illumina short read data. We observed an average of 96.98% complete BUSCO alignment and an average 99.75% remapping rate. Next, a high accuracy genome assembly would expect a very low level of homozygous SNVs from the remapping analysis and as expected we see an average of 0.00037% homozygous SNVs and 0.0073% heterozygous SNVs indicating the low error rates in these assemblies (Table 3).

Genome comparison with soybean reference genome Williams 82

IGA1008 is a soybean cultivar derived from Williams 82. Between IGA1008 and the W82_v4 assemblies, we identified 0.38 million SNVs, 0.14 million small indels, 3,203 large indels (>100 bp), 255 translocation-like and 135 inversion-like events. These events do not border or cross assembly gaps and may represent genomic variations between the two W82 lines.

Gene set completeness assessment

The numbers of protein-coding genes annotated for these cultivars were highly similar, ranging between 57,286 and 58,392 genes. We used BUSCO to evaluate the completion of our gene annotation and found that over 96% of the 1440 genes were found completely (Table 4).

Structural variation assessment

IGV92 was used to visually inspect many structural variations. We also examined known structural variations for their presence in the 8 genomes and our data confirmed the 40Kb Williams 82-specific insertion on Chromosome 15 (Supplementary Fig. 1)7,26, the I-locus inversion event (Supplementary Fig. 2)26 and the large deletion (~15Kb) affecting the E3 gene (Supplementary Fig. 3)27.

Usage Notes

Identifying variations in known genes

The resources generated in this dataset allows one to search for genomic variations in genes of interest. For instance, we were able to identify nonsense mutations in the E2 gene as well as frameshift indels in J and FT1b (Supplementary Fig. 3). These resources, such as the indel found in FT1b, has not been characterized before and provide candidate alleles for further soybean research.

PAV gene screening

As more soybean genomic resources become available, one can take a pan-genome approach to identify genomic variations that are unique or shared between different soybean accessions. As a demonstration, we included 5 additional soybean genomes (ZH1328, W0526, Lee3, PI4834633, W823) to identify genomic variations that are shared in a subset of the genomes (Fig. 2). These 5 genomes were chosen for their genome assembly quality, which were improved using sequencing technologies with longer reads. We identified 60 genes that were found in 3 wild soybeans but missing or truncated in the other 10 cultivated soybeans and 185 genes found missing or truncated in all 3 wild soybeans (Supplementary Table 3). Analyses such as this could generate additional resources to further soybean research and crop improvement.

Fig. 2
figure 2

Genomic variations of 12 soybeans compared to IGA1008 genome. The rings are in the following order from outer-most to inner-most: IGA1008 chromosomes (navy blue), PAV regions with dark red representing PV and dark green representing AV, a set of blue histograms represent SNP density in 400Kb window, the set of orange histograms represent small indel density in 400Kb window, the set of green histograms represent the large indel sizes, the inner-most set of dark blue highlights represent inversions. Each set depicts the variation of 12 soybeans ordered from outside to inside: IGA1001, IGA1002, IGA1003, IGA1004, IGA1005, IGA1006, IGA1007, ZH13, W05, Lee, PI483463, W82_v4.

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