Published online : 9 August 2022 Article Outline
Data Release
First De novo whole genome sequencing and assembly of mutant Dendrobium hybrid cultivar ‘Emma White’
Rubina Sherpa
† Rubina Sherpa
Department of Botany, Annasaheb Awate College, Manchar, Ambegoan 410503, Maharashtra, India
ICAR-National Research Centre on Orchids, Pakyong 737106, Sikkim, India
ICAR-National Research Centre on Orchids, Pakyong 737106, Sikkim, India
Find this author on Google Scholar
Find this author on PubMedRamgopal Devadas
*† Ramgopal Devadas
ICAR-National Research Centre on Orchids, Pakyong 737106, Sikkim, India
Corresponding author. E-mail: ramgopal.devadas@icar.gov.in
Penna Suprasanna
Penna Suprasanna
Nuclear Agriculture and Biotechnology Division, BARC, Mumbai 400085, Maharashtra, India
Sadashiv Narayan Bolbhat
Sadashiv Narayan Bolbhat
Department of Botany, Annasaheb Awate College, Manchar, Ambegoan 410503, Maharashtra, India
Tukaram Dayaram Nikam
Tukaram Dayaram Nikam
Savitribai Phule Pune University, Pune 411007, Maharashtra, India
564
96
Cite this article as...
Rubina Sherpa, Ramgopal Devadas, Penna Suprasanna, Sadashiv Narayan Bolbhat, Tukaram Dayaram Nikam, First
Article Information
Journal title: Gigabyte
Publisher name: GigaScience Press
Publisher location: Sha Tin, New Territories, Hong Kong SAR
Received: 05 February 2022
Accepted: 05 August 2022
Published: 9 August 2022
Preprint submitted to: https://doi.org/10.1101/2022.06.25.497579
Copyright © The Author(s) 2022.
https://creativecommons.org/licenses/by/4.0/
Data description
Background
The genus Dendrobium belongs to the tribe Podochileae and the subtribe Dendrobiinae [1]. There are about 1200 species in the genus Dendrobium, distributed throughout Southeast Asia and the Southwest Pacific islands. Dendrobium has a genome size (1C) of 0.75–5.85 pg [2] with a diploid chromosome number of 38 [3]. Dendrobium hybrids are orchids with high commercial value, and high medicinal demand and potential. Seventy percent of Dendrobiums are exported from Thailand, with a global value of US$63.6 billion [4]. They are the second best-selling potted flowering plants in the USA [5]. Scope for breeding novel Dendrobiums is limited owing to the narrow genetic makeup of hybrids from Dendrobium phalaenopsis [6], which is geographically native to Australia. There are also intersectional cross-incompatibility issues with transferring favourable genes [7]. Reverse genetics through target induced local lesions in genomics (TILLING) strategies could offer a rapid solution to trait improvement through mutation plant breeding [8].
Dendrobium nobile, known as the ‘noble orchid’, is the official state flower of Sikkim, India [9]. Its complete chloroplast genome was recently deciphered [10], and several functional genomics studies in Dendrobium have uncovered the biosynthetic pathways of alkaloids with medicinal uses [11, 12]. Given the large number of species in Dendrobium, DNA barcoding systems have been developed and tested as conservation and authentication tools [13, 14]. However, whole genome sequencing and assembly has been conducted in only four Dendrobium species of medicinal economic value [15, 16], and only using National Center for Biotechnology Information (NCBI) resources. This limits our current understanding of phylogenetic diversity among species and their relationships at the inter- and intraspecific level, and the subsequent use of this knowledge in crop improvement programmes.
Context
So far, there have been no reports of a sequence assembly for Dendrobium hybrid cultivars (NCBI:txid136990) or mutants [17]. We have applied gamma radiation to induce mutations leading to new variability for orchid genetic improvement. We chose a popular and highly adaptable Dendrobium hybrid cultivar, ‘Emma White’, which is derived from a complex cross made through a series of hybridization events using five Dendrobium species: Dendrobium phalaenopsis (six times), Dendrobium tokai (once), Dendrobium stratiotes (once), Dendrobium gouldii (twice) and Dendrobium lineale (once) as parents in pedigree between 1938 and 2006. It was developed by T Orchids, Malaysia, and was registered with the Royal Horticultural Society in 2006 [18].
The Dendrobium ‘Emma White’ hybrid cultivar is a highly cross-compatible variety when used as the female parent in hybridization programmes [7]. It easily responds to in vitro studies compared with other hybrids [19], and was used as one parent to develop a new Dendrobium breeding line, NRCO-42, which is registered with the Indian Council of Agricultural Research (ICAR) National Bureau of Plant Genetic Resources, India (accession number INGR 10073) [20]. For the first time, here we present the draft genome sequence of a gamma-induced mutant of Dendrobium hybrid cultivar ‘Emma White’ (Figure 1). This will be a valuable resource to assist with genetic improvement through future TILLING strategies.
Figure 1.
Early flowering mutant lines (10 Gy) of Dendrobium hybrid cultivar ‘Emma White’. (a) 10 Gy/08 (b) 10 Gy/27 (c) 10 Gy/41 early flowering mutant lines. (d) Comparison of control plants with flowering mutant plants.
Methods
Sampling and DNA preparation
Protocorm-like bodies (PLBs) of ‘Emma White’ hybrid plants were irradiated with gamma radiation at 10–40 Gy to induce random mutations at 32.54 Gy/min using 60Co gamma irradiator (Gamma Chamber 5000) at the Bhabha Atomic Research Centre, Mumbai, following standard protocols [21]. PLBs were cultured in vitro up to M1V5 generation and plantlets were raised from 10, 20 and 40 Gy. Subsequently, all surviving plantlets generated were moved to harden off, then were grown in polyhouse conditions for phenotypic evaluation. Early flowering mutants were identified among 10 Gy plants with several positive traits: plant height, pseudostem length, leaf number, leaf size and spikes during flowering, when there was no or delayed flowering, as in the case of control, 20 Gy and 40 Gy mutant plants.
Genomic DNA was isolated using the CTAB method [22] from 10 mg of fresh leaves of the first mutant plants during flowering. DNA sequencing libraries were prepared using a DNA library preparation kit (NEB NextUltra) and validated for quality using Agilent Tapestation. DNA fragmentation was performed according to the manufacturer’s instructions to produce fragments with an average length of 150 bp, followed by 5′ and 3′ adaptor ligation. Paired-end sequencing was performed using the Illumina HiSeqX10 platform (RRID:SCR_016385). Raw reads were used for de novo assembly using MaSuRCA (v.4.0.3; RRID:SCR_010691) with default parameters [23]. Adapter Removal (v.2) [24] was used to get rid of the adapters, low quality reads and bases. Assembly statistics were made using QUAST (v.5.2.2; RRID:SCR_001228) [25], and levels of conserved genes were generated using BUSCO (Benchmarking Universal Single Copy Orthologs; v.5.2.2; RRID:SCR_015008) [26]. The chromosome-based assembly for Dendrobium huoshanense [27, 28] was used for RagTag scaffolding using RagTag (v.2.1.0) [29] with default parameters. We also ran BUSCO on the scaffolded fasta file using the viridiplantae_odb10 lineage dataset to evaluate the assembled scaffold quality and make comparisons (Table 1).
Table 1
BUSCO results of mutant Dendrobium hybrid cultivar and RagTag scaffolded assembly.
| BUSCO values | γ mutant assembly | Scaffold assembly | ||
|---|---|---|---|---|
| Number | (%) | Number | (%) | |
| Complete BUSCOs | 286 | 16.60 | 312 | 73.4 |
| Complete and single-copy BUSCOs | 249 | 15.43 | 306 | 72.0 |
| Complete and duplicated BUSCOs | 19 | 1.18 | 6 | 1.4 |
| Fragmented BUSCOs | 433 | 26.83 | 86 | 20.2 |
| Missing BUSCOs | 913 | 56.57 | 27 | 6.4 |
| Total BUSCO groups searched | 1,614 | 100 | 425 | 100 |
Simple sequence repeats (SSRs) of each scaffold were identified using MISA (v.2.1; RRID:SCR_010765) script [30], and primer design was done on the predicted SSRs using primer3 (v.2.3.6) with default parameters [31]. MaSuRCA (v.4.0.3; RRID:SCR_010691) assembled contigs were used to run the gene prediction model using AUGUSTUS (RRID:SCR_008417) [32]. Predicted genes were compared against the Uniprot database (RRID:SCR_002380) [33] using BLASTX (RRID:SCR_001652) [34] with an e-value cut-off of 10−3 to identify potential genes governing different pathways. The best BLASTX hit based on query coverage, identity, similarity score and description of each gene was filtered out by the sequencing provider using custom python scripts and gene ontology was assigned.
Data validation and quality control
Paired-end sequencing using the Illumina HiSeqX10 sequencing platform generated 17× genome coverage with 79,792,942 reads (150 bp) for a 10-Gy/46 gamma-irradiated mutant line. On the basis of data from the NCBI Sequence Read Archive (SRA; accession number SRR16008784), this hybrid taxonomically matched 8.30% with its closest species Dendrobium catenatum, followed by Phalaenopsis equestris at 0.55%. The resulting genome assembly was 678,650,699 bp long, with a total of 635,396 contigs, with the longest being 30,571 bp and shortest being 300 bp, and a mean value of 1068 bp. The N50 value was 1423, GC content was 32.48%, and there were 447,500 contigs (Table 2).
Table 2
Genome assembly statistics of the gamma-irradiated Dendrobium hybrid mutant and RagTag scaffolding.
| Assembly | γ mutant (Dendrobium hybrid) [35] | Dendrobium catenatum [36] | Dendrobium huoshanense [27] | RagTag scaffold of γ mutant (RagTag.Scaffold) |
|---|---|---|---|---|
| #contigs (≥0 bp) | 635,396 | 286,396 | 2256 | 549,354 |
| #contigs (≥1000 bp) | 213,573 | 29,592 | 2256 | 163,119 |
| #contigs (≥5000 bp) | 8,302 | 3,709 | 1279 | 5190 |
| #contigs (≥10,000 bp) | 625 | 2,523 | 907 | 370 |
| #contigs (≥25,000 bp) | 7 | 1,684 | 448 | 29 |
| #contigs (≥50,000 bp) | 0 | 1,401 | 145 | 20 |
| Total length (≥0 bp) | 678,650,699 | 1,104,259,548 | 1,284,285,095 | 687,254,899 |
| Total length (≥1000 bp) | 439,221,924 | 1,016,149,702 | 1,284,285,095 | 471,383,498 |
| Total length (≥5000 bp) | 57,139,593 | 973,286,060 | 1,282,134,848 | 184,746,937 |
| Total length (≥10,000 bp) | 7,915,569 | 964,906,549 | 1,279,530,669 | 154,097,582 |
| Total length (≥25,000 bp) | 194,559 | 952,168,543 | 1,272,192,375 | 149,748,116 |
| Total length (≥50,000 bp) | 0 | 942,392,114 | 1,262,665,926 | 149,476,984 |
| Number of contigs | 447,500 | 64,087 | 2256 | 369,938 |
| Largest contig (bp) | 30,571 | 33,291,853 | 100,197,051 | 18,000,059 |
| Total length (bp) | 604,787,319 | 1,040,039,458 | 1,284,285,095 | 616,868,961 |
| GC (%) | 33.48 | 34.61 | 35.73 | 33.49 |
| N50 | 1423 | 1,149,703 | 71,787,458 | 2096 |
| N75 | 949 | 434,049 | 52,753,504 | 1039 |
| L50 | 105,200 | 184 | 8 | 46,924 |
| L75 | 228,327 | 553 | 13 | 154,552 |
| #Ns per 100 Kbp | 0.00 | 4167.30 | 123.03 | 1394.82 |
RagTag scaffolding of the mutant assembly, based on the reference genome of Dendrobium huoshanense, contained a total length of 687,254,899 bp; an increase of 8,604,200 bp with an N50 value of 2096 (Table 2). The final largest contig length of the RagTag scaffolded assembly increased to 18,000,059 bp from 30,571 bp, with increased N’s per 100 kilobase pairs (Kbp) to 1394.82 from 0. When compared against the Uniprot database using BLASTX with an e-value cut off of 10−3, the 96,529 genes predicted by MaSuRCA resulted in 60,741 potential genes governing different molecular functions, cellular components and biological processes. BLAST results were filtered based on a cut-off of qcov >60% and pi identity >70% to ensure confidence of annotations.
We also identified 216,232 SSRs and designed 138,856 microsatellite primers; these will be useful for generating polymorphic differences among progenies and putative gamma-irradiated mutant lines.
Most BLASTX hits showed affinity with D. catenatum based on functional annotation of genes (Figure 2). BUSCO (v.5.2.2) analysis revealed 913 (56.57%) single-copy orthologs that do not match with any databases; this indicates a possible effect of both the genomic background of the developed hybrid cultivar, and of the gamma radiation.
Figure 2.
BLASTX hits based on functional predicted gene models for different organisms.
The complex genome of the ‘Emma White’ hybrid Dendrobium cultivar is derived from five unique and unrelated species; it has been hybridized 11 times over a period of 68 years, with selection for targeted economic trait improvement (Table 1). Low BUSCO values may be attributed to its fragmented assembly. However, the presence of genomic material from several other species of same genus (otherwise contaminant species) in the hybrid cultivar may have resulted drastic changes in the missing BUSCO values [37–39]. NCBI taxonomical data for mutant Dendrobium, based on raw sequence data, also supports the view, since it has limited synteny with its closest relative, Dendrobium catenatum, at less than 9%.
In addition, multigenome hybrid cultivars are genetically heterogeneous and have an outcrossing nature, indicating higher compatibility. For example, the outcrossing species Arabidopsis lyrata had 32,670 predicted genes, even at 8.3× DNA coverage, compared with 27,025 genes in the selfing species Arabidopsis thaliana (125 megabase pairs [Mbp]), which diverged 10 million years ago [40] because of genomic loss and rearrangement. In a similar way, these novel Dendrobium hybrid cultivars have a distinct genome, because of introgression from other wild species chosen by plant breeders to create new genetic variations in a short space of time compared with evolutionary changes. It can also be attributed to deletions, mostly noncoding DNA and transposons, and the presence of a highly mutagenized background with severe developmental abnormalities; apart from presence of unclustered genes [41].
Reuse potential
The mutant Dendrobium hybrid sequencing and genome assembly presented here can be adopted as a primary reference genome, as well as complementing existing conventional Dendrobium species already in the public domain. Studies of induced mutants allow rapid discovery of new alleles at low cost using high throughput TILLING [42]. As evident from other crops [43, 44], this is especially true in vegetatively propagated Dendrobium hybrids to obtain high-density mutations using gamma radiation mutation breeding. These results provide a baseline for further research on the molecular understanding of desired traits in mutant germplasm, and to develop genomic resources for orchid improvement.
Data Availability
The de novo whole genome sequence of the gamma-irradiated mutant Dendrobium hybrid cultivar ‘Emma White’ (10 Gy) was deposited with the NCBI with SRA accession number SRR16008784 and Genbank assembly accession GCA_021234465.1 [35]. and also available in the public domain via BioProject ID PRJNA763052. Additional data is available in the GigaScience GigaDB repository [45].
Declarations
List of abbreviations
bp: base pair; BUSCO: Benchmarking Universal Single Copy Orthologs; ICAR: Indian Council of Agricultural Research; Kbp: kilobase pair; Mbp: megabase pair; NCBI: National Center for Biotechnology Information; SRA: Sequence Read Archive; SSR: single sequence repeat; TILLING: target-induced local lesions in genomics.
Ethical approval
Not applicable.
Consent for publication
Not applicable.
Competing Interests
The authors declare that they have no competing interests.
Funding
RD is funded by a research grant from the Board of Research in Nuclear Sciences, Government of India (Ref: 35/14/22/2016-BRNS/35061).
Authors’ contributions
RS conducted the gamma radiation experiment, mutant plant development and genome analysis. RD prepared the concept and research formulation of the project and wrote the manuscript. PS assisted with radiation facilities and edited the manuscript. SNB and TKN supervised the work and interpreted the data. All authors read and approved the final version of the manuscript.
Acknowledgements
The authors would like to acknowledge the Nuclear Agriculture and Biotechnology Division of the Babha Atomic Research Centre, Government of India, for providing radiation facilities. The authors are thankful to the Director of Indian Centre for Agricultural Research–National Research Centre on Orchids for providing institutional support. We also thank M/s AgriGenome Labs Private Limited, Kerala, India for the sequencing work.
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