A chromosome-scale genome of the Korean cultivar Sesamum indicumvar. Goenbaek provides insight into its oil-seed quality and health-promoting gene repertoire

doi:10.21203/rs.3.rs-4887813/v1

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A chromosome-scale genome of the Korean cultivar Sesamum indicumvar. Goenbaek provides insight into its oil-seed quality and health-promoting gene repertoire

https://doi.org/10.21203/rs.3.rs-4887813/v1

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Sesamum indicum seeds are omnipresent in Korean dietary habits as food decorations, and the oil is used in traditional medicine due to its natural antioxidant content. Although genomic resources are available mainly from Chinese and Indian genotypes, the lack of Korean genomic resources hampers the design of novel genotypes with valuable agronomic importance as well as food and health-enhancing properties. This study aimed to generate a high-quality chromosome-level genome of the Korean cultivar Goenbaek, which is rich in oil, sesamin, and sesamolin. The combination of short- and long-reads and chromosome conformation capture data helped to assemble the genome into 13 chromosomes spanning 262.5 Mbp with an N50 value of 19.9 Mbp. A total of 23,539 protein-coding genes were predicted. Phylogenetic analysis placed Goenbaek on a distinct branch, suggesting geographical or adaptive patterns leading to genetic diversity. Goenbaek exhibited a set of 1,171 species-specific gene families enriched in lipid metabolism and biosynthesis, fatty acid metabolism, and phenylpropanoid biosynthesis, which confirmed its rich oil content. In addition, health-beneficial genes related to sphingolipid and selenocompound metabolism were also identified. The new reference-grade genome will serve as a reference for the Korean genomics-assisted sesame breeding program for the improvement of agronomic traits and for the investigation of oil and specialized metabolites.

Bioinformatics

Agronomy

Plant Molecular Biology and Genetics

Epigenetics & Genomics

Sesamum indicum

comparative genomics

oil crop

lignans

sesame breeding

specialized metabolites

Sesamum indicum L. is one of the major oil crops traditionally used in Korean food culture as a cooking oil, seasoning or decoration food [1]. This member of the mint family harbors valuable nutritional and health benefits for humans, mainly due to its lignans (sesamin, sesamolin, sesaminol and sesamol). According to a recent Korean nutritional and health survey, the daily intake of lignan from sesame oil is up to 77%, with 18.39 mg and 13.26 mg per person for males and females, respectively [2].

In the last decade, tremendous effort has led to the use of this orphan crop in the genomics era [3]. The initial genome was generated using BAC cloning and short-read approaches with the Yuzhi11 cultivar [4]. Moreover, the Oil Crop Research Institute (OCRI) generated a contig-level assembly [5] that was subsequently updated into linkage groups [6]. The availability of the reference Chinese cultivar Zhongzhi13 facilitated the identification and validation of molecular markers related to seed coat color, plant height [6], seed yield components [7, 8], drought and salinity stress [9, 10], root biomass development [11], and waterlogging stress [12].

Moreover, a pangenome was constructed based on one Indian cultivar (S. indicum cv. Swetha) and four Chinese genotypes, including two cultivars (S. indicum var. Zhongzhi13 and S. indicum var. Yuzhi11) and two landraces (S. indicum cv. Baizhima and S. indicum cv. Mishuozhima) [13]. The strategy employed for pangenome construction relied on reference-based assembly with Zhongzhi13 as a backbone representative. Therefore, the need for reference-free high-quality genome resources may help to gain insights into the variability among genotypes relative to the traits of interest.

The Korean modern cultivar Goenbaek exhibited three capsules per node, an unbranched pattern, high seed yield characteristics (1.6 tons per hectare), oil (50.2% of crude fat) and lignan-enriched contents (sesamin: 3.96 mg/g, sesamolin: 2.57 mg/g), and resistance to sesame Phytophthora blight disease [14, 15]. A high-quality genome will drastically improve ongoing Korean sesame breeding activities for oil, lignans [16], and disease resistance [15]. Thus, a chromosome-grade genome assembly of this cultivar was constructed to accelerate the genomics-assisted sesame breeding program.

Plant material resources

Formal identification of the plant material was carried out by Sungup Kim, a sesame breeder who developed S. indicum var. Goenbaek [14]. A standard material transfer agreement (SMTA) was obtained for the use of S. indicum var. Goenbaek, involving the National Institute of Crop Sciences and the Korean Genebank Center, both of which are under the umbrella of the Rural Development Administration of South Korea. General information is available on the English version of the Korean Genebank Center website (https://genebank.rda.go.kr/eng/pla/pds/SimpleSearchList.do;jsessionid=NcFzrBPgDS1ViVxtAuJSlMhYuYVnrIKWZz1sVEiSfntmNP11nbekESyZ9ZrOKMlW.NAAS-AP2-mw2_servlet_engine1?isParcelOut=&radioChkTxt=&selectMenuNo=&sEngvalidspname=&sItnoS=326979&sItnoE=&sVarietyNm=&sScientificNm=&sSmpStatus=&sCountry=&recordCountPerPage=20&radioChk=B). Subsequently, a voucher specimen was deposited at the Korean Genebank Center with the GenBank code IT326979 as of June 27, 2019 (https://genebank.rda.go.kr/simpleSearchView.do?passportnum=519186).

Tissue sampling, DNA and RNA extraction

Young leaves (from 15-day-old plants) of S. indicum var. Goenbaek were collected, flash-frozen in liquid nitrogen and stored at -80°C prior to extraction. The DNA was extracted based on a modified CTAB protocol [17]. For RNA extraction, samples from the roots, stems, leaves and apical meristem were extracted using an RNeasy^® Plant Mini Kit (Qiagen, Germany). All the samples were checked for quality using a Nanodrop ND 1000 (Thermo Fisher, Waltham, MA, USA) and stored at -80°C prior to sequencing.

Short-read sequencing

After a second quality check with a Qubit fluorometer (Thermo Fisher, Waltham, MA, USA), 1 µg of genomic DNA was sheared to ~ 550 bp fragments. Then, library preparation was performed with a TruSeq Nano DNA Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer's protocol. Using the NovaSeq 6000 System (Illumina, San Diego, CA, USA), 100 bp paired-end sequencing data were generated. (Supplementary Table S1).

RNA sequencing library preparation was performed using an Illumina TruSeq RNA sample preparation kit following the manufacturer's instructions. Afterwards, 151 bp paired-end sequencing was performed using the NovaSeq 6000 platform for seeds, roots, apical meristems, stems and leaf organs (Supplementary Table S2). All short-read data were checked for quality using FastQC v0.11.2 [18] and trimmed with Trimmomatic v0.36 [19] before downstream analyses.

Genome size estimation

Using the short-read data, a k-mer-based genome size estimation was performed with GenomeScope v.2.0 [20] tools with the k-mer depth setting k = 19. The estimated genome size was 321 Mbp (Supplementary Figure S1). Based on this indicative genome size information, we conducted a subsequent PacBio long-read sequence.

PacBio long-read sequencing and initial assembly

Using high-molecular-weight genomic DNA, an ~ 20 kb SMRTbell library was constructed following the protocol of SMRTbell™ Libraries (Pacific Biosciences, Melon Parl, CA, USA). Sequencing was performed on a PacBio^® Sequel I system (PacBio CLR) with five single-molecule real-time (SMRT^®) cells using P6-C4 chemistry.

Initial assembly was performed using the FALCON package v0.7 [21]. The resulting primary assembly was error corrected with short-read paired-end data following the approach of Kang et al. [22]. Briefly, after mapping the short-read data onto the assembly using the Burrows–Wheeler Aligner (BWA) BWA-MEM algorithm (version 0.7.12) [23], the HaplotypeCaller and FastaAlternateReferenceMaker modules from the Genome Analysis Toolkit (GATK) ver. 3.54 [24] were employed to refine the base accuracy with default parameters. Furthermore, a polishing step was performed with Pilon v.1.24 [25].

Hi-C sequencing and scaffolding

To scaffold the long-read-based assembly into pseudomolecules, one Hi-C proximity ligation library was prepared using Dovetail^™’s Hi-C Kit v.1.0. The restriction enzyme DpnII was used. The prepared library was shipped to Dovetails Genomics (Scotts Valley, California, USA) for sequencing (NCBI SRA accession: SRR18210364) and scaffolding via Dovetail’s HiRise^™ scaffolding pipeline [26].

Genome assembly quality assessment

The reference-free assembly quality metric LTR assembly index (LAI) was calculated using LTR Retriever v.2.9.0 [27]. In addition, genome assembly completeness was evaluated with BUSCO v.5.2.2-0 [28] and the Embryophyta odb10 dataset (accessed 15th September 2021). The assembly contiguity was checked with QUAST-LG v.5.2.2-0 [29]. In addition, the MUMmer v4.0.0 package [30] was used to evaluate the concordance of the genome structure of the assembly with that of the reference Zhongzhi13.

Chromosome assignment structural and functional genome annotation

We assigned the chromosomes to the generated chromosomes by aligning the Hi-C assembly onto Zhongzhi13 with the nucmer package of MUMmer v4.0.0 [30] Repeat analysis was performed using the RepeatModeler v. 1.0.8 (http://www.repeatmasker.org/RepeatModeler/) and RepeatMasker v. 4.0.5 (http://www.repeatmasker.org) tools.

Noncoding RNAs, including tRNAs, rRNAs, miRNAs, and snRNAs, were identified using Infernal v1.1.4 [31] with the Rfam database [32] (accessed on the 24th, December 2021). The cross-validation of tRNA and rRNA was conducted with tRNAscan-SE v2.0.8[33] and Barrnap v0.9, [34]respectively.

The ab initio, evidence-based and homology-based annotation pipelines are illustrated in supplementary Figure S2. Briefly, a genome-guided transcriptome assembly was performed by mapping trimmed RNA-seq data onto the softmasked genome assembly using TopHat2 [35]. The mapped reads were subsequently subjected to StringTie to construct Goenbaek transcripts [36]. The resulting transcript assembly was used to train the BRAKER v1.11 [37] annotation pipeline, which uses AUGUSTUS [38] and GeneMark-ET [39] for gene prediction.

In parallel, Exonerate v2.2.0 [40] was used to refine the coding gene structure and predict near-exact intron/exon boundaries via the hard-masked genome assembly. Both evidence and ab initio annotations were integrated into EVidenceModeler (EVM)[41] to generate the gene models.

Functional annotation was performed via BLASTp (E-value: 1e-5) against Plant RefSeq, UniProt, TAIR and the National Center for Biotechnology Information (NCBI) nonredundant (nr) protein databases. Protein domains were inferred by searching against the Pfam, GO and KEGG databases using InterProScan ver. 5.34-73.0 [42] and Blast2GO Command Line ver. 1.4.1 [43]. The resulting annotations were manually curated based on the available datasets and by comparison with the reference genome Zhonghi13.

Fluorescence In Situ Hybridization (FISH)

The plant samples were prepared following the approach of Pellerin et al. [44]. Two- to three-day-old healthy root tips from seedlings grown at 25°C were sampled and pretreated with 2 mM 8-hydroxyquinoline for five hours at 12°C. The pretreated tips were fixed in Carnoy's solution for two hours at room temperature and stored in 70% ethanol until use. Somatic chromosome spread preparation was subsequently performed based on the protocol of Waminal et al. [44]. Briefly, the root tips were dissected and digested with an enzymatic solution composed of 2% cellulase and 1% pectolyase at 37°C for 90 minutes. The suspension was then washed in 70%, 80% and 90% ethanol successively to ensure dehydradation.

FISH was carried out as described by Peniton et al. [45]. Prelabeled oligo probes for 5S rDNA, 45S rDNA and Arabidopsis-type telomeric repeats were added to the mixture, which was subsequently denatured (for 5 minutes at 80°C) or hybridized (for 30 minutes at room temperature), followed by a stringent washing step.

The slides were then visualized under an Olympus BX53 fluorescence microscope with an integrated CCD camera (Leica DFC365FS; Leica Microsystems, Inc., Germany). The images were processed in CytoVision^® software version 7.2 (Leica Microsystems, Germany) and edited in Adobe Photoshop CS6.

Comparative genomics and phylogenetic construction

To identify unique genes specific to Goenbaek, we retrieved the protein sequences of S. indicum var. Zhongzhi13, S. indicum var. Yuzhi11, S. indicum cv. Baizhima, S. indicum cv. Mishuozhima, and S. indicum var. Swetha from the Oil Crops Research Institute (OCRI) pangenome repository (http://www.sesame-bioinfo.org/pangenome, accessed 15th, November, 2018). By combining the newly generated Goenbaek protein file with the abovementioned dataset, we inferred the gene clustering using OrthoFinder v.2.3.12 [46]. Afterwards, we used the STAG [47] algorithm and STRIDE [48] implemented in OrthoFinder v.2.3.12 [46] to infer and root the phylogenetic tree, respectively.

Two datasets were used for orthology and phylogenetic analyses. The first dataset included only Sesamum species, while the second included closely related species, i.e., Perilla frutescens var. frutescens (PF40) (GenBank: QFCC00000000.2), Perilla citriodora PC002 (GenBank: QIYW00000000.2), PC099 (GenBank: SDAM00000000.2) [49], Mimulus guttatus (GenBank: APLE00000000.1) [50], Solanum lycopersicum (GenBank: AEKE00000000.3) [51] and Arabidopsis thaliana (GenBank: GCA_000001735.2) [52], as an outgroup. Using Blast2GO v.5.2.5 GUI [43], we predicted the functional attributes of the Goenbaek-specific genes to determine the key variety-specific biological functions.

Assembly-based structural variant calling

To identify structural variants, Goenbaek was used to align the Zhongzhi13 reference genome using the nucmer module from the MUMmer package v4.0.0 [30] with the following setting: -maxmatch -l 100 -c 500. Afterwards, structural variants, including insertions, deletions, repeat expansions, repeat contractions, tandem expansions, and tandem contractions, were detected using the Assemblytics web server [53].

Identification of late embryogenesis abundant (LEA) genes

Candidate LEA genes representing one of the protective gene repertoires against desiccation or osmotic stresses [54] were investigated in the Goenbaek genome using hmmsearch with the following settings: E 1e-5 and domE 1e-5. The PFAMs of the different classes of LEA genes, namely, Dehydrin (PF00257.21), LEA1 (PF03760.17), LEA2 (PF03168.15), LEA3 (PF03242.15), LEA4 (PF02987.18), LEA5 (PF00477.19), LEA6 (PF10714.11), and SMP (PF04927.14), were retrieved from the Pfam website (http://pfam.xfam.org/). Candidate LEA genes were screened for LEA domains using the PfamScan command line tool v1.6 [55]. The conserved domain of each gene was double-checked with the help of the InterProScan web server (https://www.ebi.ac.uk/interpro/search/sequence/).

The physicochemical properties and subcellular localization of the proteins were assessed with ProtParam (http://web.ExPASy.org/protparam/) [56] and WoLF PSORT (https://wolfpsort.hgc.jp/) web-servers [57].

To assess the phylogenetic relationships of sesame LEA genes with well-known LEA genes from Oryza sativa [58] and A. thaliana [54], a maximum likelihood phylogenetic tree inference was performed using IQ-TREE v1.6.12 [59] with 1,000 bootstrap iterations after multiple sequence alignment and poorly aligned region trimming steps were performed with MAFFT v.7.464-0 [60] and trimAl v.1.4.1 [61], respectively. In addition, the expression levels of LEA genes in Goenbaek organs were assessed using HISAT2 v2.2.1 [62] for mapping the RNA-seq data and the FeatureCounts module from the Subread v. 2.0.1 package [63] for determining gene abundance. The expression pattern within the organs was determined using pheatmap v.1.0.12 [64] R package following Ward’s clustering method (clustering_method = “ward. D”) and gene-centered scaling (scale = “row”).

FISH analysis and high-quality chromosomal-level genome assembly

Prior to sequencing, we carried out a cytogenetic investigation through FISH karyotyping to ensure the chromosome number of the plant material. As expected (Fig. 1A, B), a total of 13 pairs of chromosomes (2n = 2x = 26) were revealed (Fig. 1C, D, E), which is consistent with previous karyotyping of the Chinese cultivar Yuzhi11 [4] and the resulting Hi-C contact map (Fig. 1B).

A total of 29.11 Gbp (90.4-fold) was generated from the long-read sequencing (Supplementary Table S3). Initial assembly resulted in 433 contigs spanning 282 Mbp with an N50 of 2.5 Mbp (Supplementary Table S4). Contig anchoring with chromosome conformation capture via Hi-C data drastically improved the contiguity of the draft assembly by 7.6-fold. A total of 249 sequences were identified with a high signal contact map (Supplementary Table S4). The identified 13 chromosome set size was 282 Mbp, with an N50 value of 19.9 Mbp (Supplementary Table S5). By comparing the chromosomal assembly set of Goenbaek and the reference gene Zhongzhi13 (Supplementary Table S5), a substantial improvement was achieved at the gap level. In fact, 183 gaps were counted for Goenbaek, while 8,906 gaps were counted for Zhonghi13. Several metrics were used to check the quality of our assembly, including assembly contiguity (NG50) (Supplementary Figure S3), assembly completeness (BUSCO) (Supplementary Table S6), assembly continuity (LTR Assembly Index) (Supplementary Figure S4) and collinearity (whole-genome alignment with the MUMmer package onto Zhongzhi13) (Supplementary Figure S5). Although the contiguity was similar (Supplementary Table S5, Supplementary Figure S3), the BUSCO analysis performed on the assemblies from the Embryophyta odb10 dataset revealed better coverage of single-copy orthologs in Goenbaek (92.8%) than in Zhongzhi13 (24.5%) (Supplementary Table S6). However, the assessment of the genes annotated by BUSCO revealed that Zhongzhi13 had more (90.1%) single-copy gene sets than did Goenbaek (86.2%). When considering the LAI (Supplementary Figure S4), the overall value (17.47) in Goenbaek was greater than that in Zongzhi13. Similar observations were noted by comparing each paired-chromosomal region.

Overall, the quality assessment results imply that Goenbaek exhibits reliable contiguity and completeness, making it a useful resource for Korean sesame breeding programs.

Structural variants detection

One of the key determinants of genetic diversity is structural variants since they have an enormous impact on gene regulatory regions [65, 66]. In the present study, we detected 7,578 SVs, including 1,693 insertions, 1,635 deletions, 1545 repeat expansions, 1,487 repeat contractions, 1,046 tandem contractions, and 162 tandem expansions (Fig. 3A). Among the 3.9 Mbp, repeat contraction and expansion were the major contributors to SVs spanning 1.4 and 1.1 Mbp, respectively (Fig. 3B). SV fragments ranging from 50 bp to 500 bp in length were the most abundant in all the classes, representing 59.87%-95.04% of the total length. Notably, the SVs of other species have not been investigated since their assemblies were Zhongzhi13-based.

Genome annotation

The repeat sequences in the Goenbaek genome spanned approximately 128.1 Mbp, which represents 48.83% of the assembled genome (Table 1). Long terminal repeat (LTR) retrotransposons are prominent and occupy 20.2% of the genome. Among the LTRs, the Copia and Gypsy types were the most abundant, accounting for 9.1% and 7.3%, respectively. The number of repeat elements in S. indicum var. Goenbaek is greater (48.83%) than that in S. indicum var. Zhongzhi13 (28.5%) [5] and S. indicum var. Yuzhi11 (31.65%) [4] but lower than that in the close relatives Perilla frutescens (64.1%) and Perilla citriodora (56.7%) [49].

Table 1

Repeat content information for the sesame variety Goenbaek
Class	Elements	Count	Base	Percent
DNA	Uncharacterized	813	258,832	0.10%
	CMC-EnSpm	6,716	3,696,805	1.41%
	CMC-Transib	1,060	52,414	0.02%
	Crypton	586	304,925	0.12%
	Dada	316	68,115	0.03%
	En-Spm	2,809	235,867	0.09%
	Harbinger	61	9,347	0.00%
	MULE-MuDR	13,205	3,915,495	1.49%
	Mite	89	4,562	0.00%
	MuDR	377	40,420	0.02%
	PIF-Harbinger	1,320	871,778	0.33%
	Sola	1,000	487,100	0.19%
	TcMar	1,544	313,493	0.12%
	TcMar-Pogo	859	291,316	0.11%
	TcMar-Stowaway	275	35,287	0.01%
	hAT	360	89,733	0.03%
	hAT-Ac	4,696	1,346,792	0.51%
	hAT-Charlie	1,984	385,492	0.15%
	hAT-Tag1	2,366	576,646	0.22%
	hAT-Tip100	54	14,449	0.01%
Total DNA		40,490	12,998,868	4.95%
LINE	Uncharacterized	722	116,263	0.04%
	Jockey	42	17,361	0.01%
	L1	6,135	6,155,849	2.35%
	R1	214	53,257	0.02%

Table 1

*Cont.*
	R2	420	233,710	0.09%
Total LINE		7,533	6,576,440	2.51%
LTR	Uncharacterized	26,562	6,731,082	2.56%
	Caulimovirus	2,245	2,895,561	1.10%
	Copia	30,561	24,013,267	9.15%
	Gypsy	29,582	19,223,979	7.32%
	Ngaro	1,083	118,297	0.05%
Total LTR		90,033	52,982,186	20.18%
SINE	Uncharacterized	9	657	0.00%
	Alu	61	20,325	0.01%
Total SINE		70	20,982	0.01%
MobileElement		535	101,176	0.04%
Other		364	31,414	0.01%
Helitron		353	67,806	0.03%
Retroelement		140	28,681	0.01%
Unknown		194,737	55,237,760	21.04%
nonLTR		112	14,570	0.01%
Satellite		4	483	0.00%
Simple_repeat		479	115,181	0.04%
Total		334,850	128,175,547	48.83%
Total nonredundant			118,036,267	44.97%

The inference of noncoding RNA returned 841 tRNAs, 416 rRNAs, 123 miRNAs, and 134 snRNAs, spanning 62.55 Kbp, 311.85 Kbp, 16.79 Kbp, and 13.65 Kbp, respectively (Table 2). For evidence-based annotation, a total of 37.3 Gb of transcriptome data from leaves, roots, stems, apical meristems, and seeds were generated (Supplementary Table S2). Gene prediction revealed 23,539 protein-coding genes with average gene, exon, and intron lengths of 3,155 bp, 245 bp, and 472 bp, respectively. Compared to Zhongzhi13 (34,451 coding genes), the gene count was lower. However, these results are comparable to those of the Yuzhi11 and Baizhima gene sets, which exhibited 21,360 and 27,015 genes, respectively (Supplementary Table S5).

Table 2

Predicted noncoding RNA statistics
Type	Number	Total length (bp)
tRNA	841	62,549	0.024%
rRNA	416	311,851	0.119%
miRNA	123	16,785	0.006%
snRNA	134	13,645	0.005%

According to our functional annotation analysis, 91.1% of the Goenbaek genes exhibited abundant sequence similarity with Plant RefSeq sequences, while 73.9% and 84.2% sequence similarity were found between the UniProt and A. thaliana databases, respectively (Table 3).

Table 3

Functional annotation information from the predicted protein-coding genes of Goenbaek
Annotation category		Hit count	Percent(%)
BLASTP	Plant RefSeq	21,438	91.1%
	UniProt	17,389	73.9%
	Arabidopsis thaliana	19,823	84.2%
InterProScan	GO	10,968	46.6%
	KEGG	1,061	4.5%
	Pfam	17,119	72.7%
Transcriptome	mRNA-Seq read count	16,368	69.5%
Total hit		21,875	92.9%
Known		19,708	83.7%
Uncharacterized		2,027	8.6%
Hypothetical		1,804	7.7%
Total		23,539	100.0%

A domain search of the Pfam, GO and KEGG databases revealed that 72.7%, 46.6%, and 4.5%, respectively, of the predicted genes had known domains. Altogether, 83.7% of the predicted Goenbaek genes were functionally annotated based on known protein sets, while uncharacterized and hypothetical genes accounted for 8.6% and 7.7%, respectively.

Goenbaek-specific Genes Repertoire and its Phylogenetic Placement

The proteins predicted from the Goenbaek, available cultivar and landrace protein sets were compared to identify Goenbaek-specific gene families (Fig. 4A). An additional protein dataset from Lamiales species, including the Perilla, Mimulus and Solanum genera, was generated with A. thaliana as an outgroup to infer the phylogenetic position of Goenbaek (Fig. 4B, C).

The gene clustering analysis revealed a total of 9,919 gene families shared by all S. indicum species, while 1,171 gene families (Supplementary Table S7) were specific to Goenbaek (Fig. 4A). KEGG analysis revealed that the unique gene families of Goenbaek were enriched in several biological functions, including alpha-linoleic acid metabolism (ko00592), fatty acid biosynthesis (ko00061), penose and glucuronate interconversions (ko00040), glycerolipid metabolism (ko00561), galactose metabolism (ko00052), and phenylpropanoid biosynthesis (ko00940).

The abundant occurrence of lipid metabolism-related functions suggested that Goenbaek has undergone important seed quality-oriented selection. In fact, in Korea, Goenbaek has high oil and lignan contents [14]. Similar biological oil-related functions were also predicted to be unique to the other cultivars Zhongzhi13, Yuzhi11 and Swetha, while landraces exhibited an adaptive gene repertoire related to plant‒pathogen interactions, protein folding, signal transduction, protein processing in the endoplasmic reticulum, and PI3K–Akt signaling [13].

Furthermore, the unique genes identified in Goenbaek included health-promoting proteins involved in drug metabolism (ko00983), selenocompound metabolism (ko00450), sphingolipid metabolism (ko00600), and the glycosphingolipid biosynthesis–ganglio series (ko00604), indicating the presence of health-promoting genes. Notably, the specialized metabolites sesamin and sesamolin were highly expressed in the variety [14]. Sesame lignans (sesamin, sesamol, sesamolin, and sesaminol) are well known for their antimicrobial, antioxidant, and anticancer properties [58, 67, 68]. Moreover, sphingolipid metabolism and selenocompound biosynthesis have also been identified as anticancer [69] and antioxidant [70] agents, respectively.

The inferred phylogenetic tree placed Goenbaek in the Sesamum clade, which is genetically distant from the other S. indicum species (Fig. 4A, B). The tree topology was consistent with the results of Yu et al. [13], who showed that Zhongzhi13 and Yuzhi11 formed a subclade (Chinese cultivars), while the Chinese landraces (Baizhima and Mishuozima) also clustered together. The Indian and Korean cultivars are distinct from their congeners, implying geographical and/or environmental effects on genetic diversity. The observed placement of Goenbaek might be linked to its species-specific genes since the number of genes in the species-specific orthogroups was 3.5 times greater than that in Zhongzhi13 (Supplementary Table S8). This rate was drastically greater than those of Yuzhi11 (47.5 times), Baizhima (21.75 times), Mishuozhima (37.3 times), and Swetha (1.23 times), confirming the phylogenetic topology.

Genome-wide identification and phylogenetic analysis of SiLEA genes

LEA genes have been shown to be functionally correlated with the response to abiotic stress by acting as molecular shields [71] and hydradation buffers [72], interacting with cell membranes for signaling [73], exerting enzyme protection [74], or regulating phytohormones [75]. To characterize the Goenbaek LEA gene repertoire, the conserved domain of each protein, phylogenetic inference, and physical and chemical parameters, including the isoelectric point (pI), aliphatic index (AI), and grand average of hydropathicity (GRAVY), were investigated. A total of 71 LEA genes were identified and classified into six classes: LEA1, LEA2, LEA3, LEA4, LEA5, and SMP (Supplementary Table S9, Supplementary Table S10, Fig. 5A, and B). The most abundant class was LEA2, which encompassed 56 proteins, while the other classes, SMP, LEA1, LEA4, LEA3 and LEA5, contained 5, 4, 4, 1, and 1 member(s), respectively. The longest protein, SiLEA2.13 (1037 aa), had a molecular weight of 118.4 kDa, while the smallest, SiLEA5 (112 aa), had a molecular weight of 12.2 kDa.

The isoelectric points ranged from 4.38 (SiSMP4) to 10.51 (SiLEA2.18), with an average of 8.54. The following proteins, including SMP (SiSMP1, SiSMP2, SiSMP3, and SiSMP4), LEA1 (SiLEA1.2 and SiLEA1.4), LEA2 (SiLEA2.8 and SiLEA2.13; SiLEA.19 and SiLEA2.29; SiLEA2.48 and SiLEA2.49; SiLEA2.52 and SiLEA2.55; LEA4 (SiLEA4.1 and SiLEA.3; and SiLEA.4) and LEA5 (SiLEA5) members, had the highest (AI > 100) aliphatic indices. With the highest AIs, the mentioned proteins are likely to be more thermally stable (Supplementary Table S9).

Expression profile of SiLEA genes in Goenbaek

The expression profiles of different tissues (leaf, stem, seed, apical meristem, and root) (Fig. 5C) revealed that the SiLEA2 gene class was present within all organs, while the SiLEA1 genes were mostly highly expressed in seeds. Most of the SiLEA4 genes were also expressed in seeds, except for SiLEA4.2, which is also expressed in the apical meristem. The unique SiLEA5 gene was also preferentially expressed in seeds, while the SiSMP genes were expressed not only in seeds but also in apical meristems, leaves and stems. The relative presence of LEA proteins within all organs suggested that these genes contributed to plant growth and development. The specific prominence of the SiLEA2 gene in all organs suggested that Goenbaek has a rich LEA2 repertoire that can play an active role in the context of abiotic stress, including drought stress. Specifically, the relatively high expression of the LEA2 gene in roots suggested its functional role in root development [76].

As the initial part of the Korean sesame breeding program, we generated a high-quality chromosomal grade genome of the elite cultivar Goenbaek with a total of 23,539 protein-coding genes. Comparative genomics highlighted strong selection pressure from humans regarding seed quality as a source for oil and lignan production. Phylogenetic inference revealed that Goenbaek is genetically distinct from its congeners, implying geographical or adaptive-driven genetic diversity. Moreover, we investigated the LEA gene repertoire of Goenbaek, providing initial molecular resources for abiotic stress management.

Given the economic importance of sesame oil as well as its health-promoting properties, the present genomic resource offers a path for sesame breeding regarding not only routinely targeted agronomic traits but also food- and health-promoting attributes.

Supplementary files

Supplementary Fig. 1. GenomeScope profile of Sesamum indicum var. Goenbaek using Illumina short reads via k-mer prediction at k = 19

Supplementary Fig. 2. Diagrammatic representation of the genome annotation of Sesamum indicum var. Goenbaek

Supplementary Fig. 3. NG(x) chart showing the contiguity of both genome assemblies. The NG50 value is shown as a dotted line

Supplementary Fig. 4. Dot plot showing the LTR assembly index (LAI) distribution within each chromosome of Goenbaek and Zhongzhi13. The LAI values are colored as follows: red for LAI values between 0 and 10, purple for LAI values between 10 and 20, and green for LAI values greater than 20. The tracks were summed in 500 Kbp windows with a 25 Kbp step size.

Supplementary Fig. 5. MUMmer plot of the alignment of Goenbaek onto Zhongzhi13 (whole genome and from chromosome 1 to 7)

Supplementary Fig. 6. MUMmer plot of the alignment of Goenbaek onto Zhongzhi13 (from chromosome 8 to 13)

Supplementary Table 1. Short-read paired-end sequencing raw data

Supplementary Table 2. RNA-seq raw data information for Goenbaek annotation

Supplementary Table 3. PacBio sequencing-filtered read data statistics

Supplementary Table 4. Primary and Hi-C assembly statistical information;

Supplementary Table 5. Comparative statistics of 13 chromosome assemblies of varieties and landraces

Supplementary Table 6. Comparative BUSCO analysis of Goenbaek and the reference genome Zhongzhi13 using the Embryophyta odb_10 dataset

Supplementary Table 7. List of Sesamum indicum var. Goenbaek-specific protein sequences

Supplementary Table 8. Comparative statistics of 13 chromosome assemblies of varieties and landraces

Supplementary Table 9. Information on the identified late abundant embryogenesis (LEA) genes in the Sesamum indicum var. Goenbaek genome

Supplementary Table 10. Late abundant embryogenesis (LEA) gene sequences from Sesamum indicum var. Goenbaek.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Funding

This study was carried out with the support of the Cooperative Research Program for National Agricultural Genome Program (Project No. PJ01347002), Rural Development Administration, Republic of Korea.

Authors’ contributions

K.L. designed the project. Y.A.B.Z. and K.L. conducted the wet-lab experiments. Y.A.B.Z. performed the bioinformatics analyses. Y.A.B.Z. wrote the manuscript. S.K.T., Y.M., A-B. O., J-G. K., and K.L. reviewed the manuscript. All the authors read and approved the final draft of the manuscript.

Acknowledgments

The authors are grateful to the National Institute of Crop Science for plant material resource support.

Availability of data and materials

The data supporting the findings of this work are available within the paper and its supplementary information files. The genome sequence data and genome assembly have been deposited in the NCBI under the BioProject accession number PRJNA810203. The genome assembly and annotation data are also available at https://zenodo.org/record/6350881.

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The authors declare no competing interests.

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Supplementary_Tables_S1-S6
SupplementaryTableS7.xlsx
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A chromosome-scale genome of the Korean cultivar Sesamum indicumvar. Goenbaek provides insight into its oil-seed quality and health-promoting gene repertoire

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Abstract

Figures

Introduction

Materials and methods

Plant material resources

Tissue sampling, DNA and RNA extraction

Short-read sequencing

Genome size estimation

PacBio long-read sequencing and initial assembly

Hi-C sequencing and scaffolding

Genome assembly quality assessment

Chromosome assignment structural and functional genome annotation

Fluorescence In Situ Hybridization (FISH)

Comparative genomics and phylogenetic construction

Assembly-based structural variant calling

Identification of late embryogenesis abundant (LEA) genes

Results and discussion

FISH analysis and high-quality chromosomal-level genome assembly

Structural variants detection

Genome annotation

Goenbaek-specific Genes Repertoire and its Phylogenetic Placement

Genome-wide identification and phylogenetic analysis of SiLEA genes

Expression profile of SiLEA genes in Goenbaek

Conclusions

Supplementary files

Declarations

Funding

Authors’ contributions

Acknowledgments

Availability of data and materials

References

Additional Declarations

Supplementary Files

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