First De novo whole genome sequencing and assembly of mutant Dendrobium hybrid cultivar ‘Emma White’

The Dendrobium hybrid cultivar ‘Emma White’ is an ornamental, successfully commercialised orchid. We used a gamma ray-induced early flowering mutant and the Illumina HiSeqX10 sequencing platform to generate the first draft de novo whole genome sequence and assembly. The draft sequence was 678,650,699 bp in length, comprising 447,500 contigs with an N50 of 1423 and 33.48% GC content. Comparing 95,529 predicted genes against the Uniprot database revealed 60,741 potential genes governing molecular functions, biological processes and cellular components. We identified 216,232 simple sequence repeats and 138,856 microsatellite markers. Chromosome-level genome assembly of Dendrobium huoshanense was used to RagTag-scaffold available contigs of the mutant, revealing a total length of 687,254,899 bp with an N50 of 2096. The longest final contiguous length was 18,000,059 bp from 30,571 bp. BUSCO genome completeness was 93.6%. This study is valuable for investigating the mechanisms of mutation, and developing Dendrobium hybrid cultivars using mutation breeding.


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 [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.

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 60 Co 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   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.

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

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].

ETHICAL APPROVAL
Not applicable.

CONSENT FOR PUBLICATION
Not applicable.