Long-read datasets of Kayu Bawang (Azadirachta excelsa (Jack) Jacobs) from 3 different identified seed stands in Bengkulu (Sumatra), Indonesia and its phylogenetic relationships

Introduction

Azadirachta excelsa (Jack) Jacobs or known as Kayu bawang (Meliaceae) is a main local wood in Bengkulu Province that is cultivated in private forests, mainly found in North Bengkulu and Central Bengkulu Districts (Depari et al. 2015; Siahaan & Sumadi 2015). Kayu bawang has been used for carpentry wood. Historically, this wood has been a favorite one of the local community as the primary material for building. In addition, the wood is also known for its resistance to attack by destructive organisms (Krisdianto et al. 2015). Despite kayu bawang is widely used for building wood and furniture, but some references are still ambiguous with respect to its scientific naming. Some references mentioned the naming of kayu bawang tree in Bengkulu as Protium javanicum Burm F (Adfa et al. 2013). In some other references, kayu bawang is named differently such as Dysoxylum mollissimum Blume (Ishiguri et al. 2016), Azadirachta excelsa (Jack) M Jacobs (Premono & Lestari 2014). In addition, in the identified seed stand (TBT) in Bengkulu as registered under No. 002/BPSMBK/SSB/2016, No. 001/BPTH.I-3/SSB/2016, and No. 006/P2STH/SSB/2018, kayu bawang is named respectively as Azadirachta exelsa, Dysoxylum molissimum, and Melia excelsa (synonym of Azadirachta excelsa). Based on morphological identification on Herbarium Bogoriense under National Research and Innovation Agency (BRIN), kayu bawang has been confirmed as Azadirachta excelsa (Jack) Jacobs. We used three specimens from different identified seed stands, namely TBT-A, TBT-K, and TBT-S, that were collected with the specimen numbers BO1991351 (TBT-A), BO1991352;1991353;1991354 (TBT- K), and BO1991350 (TBT-S) for genome sequencing using Oxford Nanopore Technologies. The objectives of the research were to assembly long-read DNA sequences and to construct a phylogeny for verifying species identification.

Methods

Genomic DNA was extracted from kayu bawang (Azadirachta excelsa) cambium samples which were taken from three different identified seed stands namely Talang Boseng (TBT-A, -3.66890°S, 102.292122°E), Batu Ampar (TBT-K, -4.394333°S, 103.138056°E), and Penyangkak (TBT-S, -3.531333°S, 102.252861°E) in Bengkulu Province, Indonesia. The modified CTAB (cetyl trimethyl ammonium bromide) method was used to extract the genomic DNA from each A. excelsa cambium (Doyle & Doyle 1987). The DNA extract buffer contained 200 μL of 10% CTAB (CAT NO. 194004, LOT NO. QR15741, MP Biomedicals, LLC), 100 μL of 1 M Tris-HCL, 280 μL of 5M NaCl, 40 μL of 0.5 M EDTA, 100 μL of 1% PVP, 280 μL of aquades. 0.1 g cambium mashed using mortar milling was put into a 2 mL tube, 1000 μL of DNA extract buffer that had been incubated at 65°C (60 min), 40 μL of 26% PVP, and 40 μL of β-Mercaptoethanol. The mixture was homogenized using a vortex and subsequently incubated at 65°C (60 min) with rehomogenization performed every 10 min by gently flipping the tube. The cooled mixture was centrifuged for 10 min, at 10,000 rpm. The supernatant was transferred to a 1.5 mL tube, and 500 μL of chloroform: isoamyl alcohol solution (24:1) and 10 μL of phenol were added and centrifuged for 10 min, at 10,000 rpm. Separation of the mixture was done again by adding 500 μL of chloroform: isoamyl alcohol solution (24:1). The obtained supernatant was put into a 1.5 mL tube, then 500 μL cold isopropanol and 250 μL NaCl were added. The mixture was stored in a freezer at 4°C for 1 day so that the DNA precipitation process occurring. After DNA precipitation, the mixture was centrifuged for 10 min, at 10,000 rpm. The mixture was removed gradually to retrieve the DNA pellet. DNA pellet washing using 500 μL of 70% ethanol, centrifuged for 10 min, at 10,000 rpm. The DNA pellet was separated from the mixture and dried in a desiccator containing silica gel (±20 min). 50 μL of TE RNAse buffer was added to dissolve the DNA pellet. The quality of extracted genomic DNA was evaluated using 1% agarose gel electrophoresis. The quality and quantity of the extracted genomic DNA were measured using NanoPhotometer^® NP80 (IMPLEN) and Qubit 1.0 Fluorometer (Thermo Fisher Scientific) with the Qubit dsDNA BR assay kit. The library preparation of long-read sequencing was performed using Oxford Nanopore Technologies (ONT) manufactured protocol SQK-LSK109 version NBE_9065_v109_revZ_14Aug2019 with the MinION R9.4.1 flow cell (FLO-MIN106D, LOT 11001352) on a MinION Mk1C sequencer (Oxford Nanopore Technologies).

The chloroplast genome assembly for TBT-A, TBT-K, and TBT-S (Accession number DRA015537; https://ddbj.nig.ac.jp/resource/bioproject/PRJDB14301) (IPB University 2023) was performed using Galaxy Server (https://usegalaxy.eu/, RRID: SCR_006281, MIT License) v.23.1.1.dev0 following long-read data with some modifications (Wang et al. 2018). Long-read data for each sample was quality checked using NanoPlot (https://usegalaxy.eu/, RRID:SCR_024128, MIT License) v1.41.0 (De Coster et al. 2018). Those clean reads data were aligned with the reference NC_023792.1 (Azadirachta indica) using Minimap2 (https://usegalaxy.eu/, RRID:SCR_018550, MIT License) v.2.26 (Li 2018) and SAMTools (https://usegalaxy.eu/, RRID:SCR_002105, MIT License) v.2.05 (Li et al. 2009). Chloroplast reads were assembled using Canu Assembler (https://usegalaxy.eu/, RRID:SCR_015880, GPLv2 and others) v.2.1.1+galaxy0 (Koren et al. 2017) involving parameters such as technology Nanopore, estimated genome size 160k, and min read length 1000. Medaka Consensus Pipeline (https://usegalaxy.eu/, RRID:SCR_005857, Mozilla Public License 2.0) v1.7.2+galaxy1 was used to polish the assembly result (Oosterbroek et al. 2021). Geseq (https://chlorobox.mpimp-golm.mpg.de/geseq.html, RRID:SCR_017336, MIT License) (Tillich et al. 2017) was used to annotate the assembled chloroplast genome using all Meliaceae reference in NCBI RefSeq and visualized using OGDRaw (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html, RRID:SCR_017337, MIT License) in the CHLOROBOX (https://chlorobox.mpimp-golm.mpg.de/index.html, MIT License) (Greiner et al. 2019). Phylogenetic analysis was performed using MEGAX (https://www.megasoftware.net/, RRID:SCR_023471, MIT License) v10.2.2 (Kumar et al. 2018) with Maximum Likelihood method, Tamura-3 model and bootstrap value of 1000 replications. rbcL gene marker was used to construct the phylogenetic tree from TBT-A, TBT-K, and TBT-S samples (Owenia vernicocsa (DQ238063.1) as an outgroup) (Muellner et al. 2006).

Results

The library yielded 481.6 Mb for TBT-A, 597.4 Mb for TBT-K, and 853.1 Mb for TBT-S. Based on the statistics, TBT-A has 3,339 bp of N50, 3,103 bp for TBT-K, and 3,882 bp for TBT-S (Figure 1). Chloroplast draft genomes of the TBT-A sample (36% GC content) produces 58,780 bp containing 14 tRNAs and 47 encoding genes, TBT-K sample (37% GC content) produced 142,139 bp containing 4 rRNAs, 24 tRNAs, and 78 encoding genes, also TBT-S sample (36% GC content) produced 84,906 bp containing 24 tRNAs and 53 encoding genes (Table 1). Based on the constructed phylogenetic tree, Azadirachta excelsa from three identified tree stands (TBT-A, TBT-K, and TBT-S) were placed in the same group with other Azadirachta excelsa accessions (Figure 2). We can confirm that all those samples belong to Azadirachta excelsa.

Figure 1. Chloroplast draft genome of Azadirachta excelsa (TBT-A, TBT-K, and TBT-S samples).

Figure 2. Phylogenetic tree based on Maximum Likelihood method.

GenBank accession numbers are listed beside the scientific name. The bootstrap value of 1000 replications is shown next to the nodes. Azadirachta excelsa (TBT-A, TBT-K, and TBT-S samples) are marked in coloring red.

Author roles

Efratenta Katherina Depari: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Resources, Visualization, Writing-Original Draft;

Nurheni Wijayanto: Investigation, Supervision, Writing-Review & Editing;

Lina Karlinasari: Supervision, Validation, Writing-Review & Editing;

Rafi M: Resources, Supervision, Writing-Review & Editing;

Iskandar Zulkarnaen Siregar: Conceptualization, Funding Acquisition, Methodology, Resources, Supervision, Validation, Writing-Review & Editing.