The complete mitochondrial genome of Allium fistulosum L. (Amaryllidaceae)

Abstract

Allium fistulosum L. (Linnaeus, Carolus, 1753) is an aromatic vegetable with health benefits and medicinal value. In this study, the complete mitochondrial genome of A. fistulosum was determined. Circular mitochondrial DNA (mtDNA) was 382,053 bp in size, encoded 44 genes, and contained 26 protein-coding genes (PCGs), 14 tRNAs, and four rRNAs. Phylogenetic analysis of amino acid sequences of the 26 PCGs revealed that the closest relationship was between A. fistulosum and Allium cepa. The high-quality mitochondrial genomic sequences of A. fistulosum presented in this study will serve as a useful resource for a range of genetic, functional, evolutionary, and comparative genomic studies on this species of the Amaryllidaceae family.

Keywords:

• Allium fistulosum L
• mitochondrial genome
• phylogenetic analysis

Introduction

Allium fistulosum L. (Linnaeus, Carolus, 1753) is a perennial herb belonging to the family Amaryllidaceae that is widely distributed in Asia (Singh and Ramakrishna 2017). A. fistulosum is a typical spicy vegetable, is an excellent flavoring agent in dietary cooking and plays an important role in preventing human diseases (Gao et al. 2022; Tang et al. 2022).

The mitochondrial genome contains genetic and evolutionary information about the species, and its high conservation in eukaryotes makes it a useful marker for evolutionary research (Zheng et al. 2018). To date, only the complete mitochondrial genome of Allium cepa has been published in the Amaryllidaceae family (Kim et al. 2016), and there is no information on the mitochondrial genome of A. fistulosum. To our knowledge, this is the first study to report the complete mitochondrial genome of A. fistulosum.

Materials and methods

In the present study, fresh leaf sample of A. fistulosum were collected from the Beijing Vegetable Research Center of the Beijing Academy of Agricultural and Forestry Sciences (BVRC), Beijing, China (11°54′55″ E, 40°21′58″ 36 N) (Figure 1). The voucher specimen was deposited in the Germplasm Resource Bank of the BVRC under voucher number 31310 (contact person: Yongqin Wang; [email protected]). Leaf mitochondrial DNA was extracted as previously described by Chen et al. (2011). Purified DNA was sequenced on an Illumina Hiseq 4000 and PacBio, assembled in SPAdes v3.10.1 (Borgström et al. 2011). Geseq (https://chlorobox.mpimp-golm.mpg.de/geseq.html) was used to annotate the mitochondrial genome. OrganelleGenomeDRAW software (OGDRAW) was used to map the mitochondrial genome (Greiner et al. 2019). The complete mitogenome sequences of the nine species were downloaded from GenBank and aligned using the MAFFT website (Katoh and Standley 2013). Phylogenetic analyses were performed using MEGA 6 with 1000 bootstrap replicates, and the model-finding tool in MEGA 6 was used to acquire the best replacement model. During this process, maximum likelihood analysis was performed, and the cpREV model was chosen as the appropriate model for sequencing, with Indigofera tinctoria (MW448462) and Nicotiana tabacum (NC_006581) considered as outgroups. Primers for the identification of the cox2 gene were referred to Kim et al. (2013).

Figure 1. Species reference map and pictures of the growth and development process of Allium fistulosum. (A) Whole plant of A. fistulosum. A. fistulosum is a monocotyledonous plant with green tubular leaves. Pseudostems are formed by metamorphic development of leaf sheaths. The stem is extremely shortening. The root system is fibrous roots with strong regenerative ability. (B) Seeds. The seeds are black, shield-shaped, with a raised, angular back and a semicircular abdomen; (C) Seedling. The seedling is of elongated tubular leaf young leaves that are light green. (D) Unopened flower bud. The unopened buds are covered with a membrane; E: Blooming bud. The apical umbellate florets are spherically clustered and arranged radially. These photos were taken by the author of Jiayi Xing.

Results

The mitochondrial genome of A. fistulosum was sequenced to an average depth of 1207 X (supplemental Figure S1), indicating that it could be used for subsequent bioinformatic analysis of the mitochondrial genome. The total mitochondrial genome length of A. fistulosum was 382,053 bp (GenBank accession number OL347690), with a GC content of 44.98%. It consisted of 26 protein-coding genes (PCGs) (atp1, atp4, atp6, atp9, ccmB, ccmC, ccmFN1, ccmFN2, ccmFC, cob, cox1, cox2, cox3, matR, mttB, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, nad7, nad9, and two rps12), 14 tRNA genes, and 4 rRNA genes (Figure 2). All PCGs used ATG as the start codon, except for cox1, nad1, nad4L, and rps12, for which the start codons were GTG, ACG, ACG and ACT, respectively. The 14 tRNA-coding genes ranged in size from 67 bp to 83 bp (Table 1). With regard to stop codons, three PCGs (atp6, atp9, and ccmFC) were terminated with CAA or CGA, whereas the remaining 23 PCGs were terminated with routine termination codons (TAA, TAG, or TGA). Gene annotation prediction results showed that the genome contained 3 cis-splicing genes (ccmFC, nad4, and nad7) and 4 trans-splicing genes (cox2, nad1, nad2, and nad5) (supplemental Figures S2 and S3). And the PCR identification result of the cox2 in Asparagus, A. cepa and A. fistulosum also showed that cox2 was trans-splicing (supplemental Figure S4), which was similar to the findings of Kim et al. (2013). Additionally, the phylogenetic analysis showed that A. fistulosum was closely related to A. cepa, followed by Asparagus officinalis (Figure 3).

Figure 2. Mitochondrial genome map of Allium fistulosum, with 26 protein-coding genes (PCGs), 14 tRNAs, and four rRNAs. The rRNAs genes were filled in red, and genes with related functions are shown in the same color. Genes that are found in the inner and outer circle are transcribed clockwise and counterclockwise, respectively. The colored parabola in the center circle represents the dispersed repeats.

Figure 3. A maximum-likelihood (ML) tree was constructed based on ten species of mitochondrial genomes (mtDNA) with 1000 bootstrap replicates, with Indigofera tinctoria (MW448462) and Nicotiana tabacum (NC_006581) as outgroups. The newly sequenced species in this study is indicated in red. The following sequences were used: A. cepa AP018390 (Shigyo et al. 2018), Asparagus officinalis NC_053642 (Sheng 2020), Cocos nucifera NC_031696 (Aljohi et al. 2016), Phoenix dactylifera NC_016740 (Fang et al. 2012), Apium leptophyllum MZ328723, Triosteum pinnatifidum NC_064333, Fragaria orientalis NC_057524 (Liu et al. 2021), Indigofera tinctoria MW448462 (Choi et al. 2021), Nicotiana tabacum NC_006581 (Sugiyama et al. 2005). NCBI accession numbers are given for Genbank sequences.

Table 1. Gene information of the mitochondrial genome of A. fistulosum.

	Position			Nucleic acid			Codon
gene	Start	End	Length (bp)	AT(%)	GC(%)	Cod-ing strand	Start	Stop	Anti-codons	Amino acid size
atp1	365329	366852	1524	54.92%	45.08%	+	ATG	TGA		507aa
atp4	223541	224104	564	58.33%	41.67%	−	ATG	TAA		187aa
atp6	191285	191998	714	61.62%	38.38%	+	ATG	CAA		237aa
atp9	364693	364917	225	55.11%	44.89%	+	ATG	CAA		74aa
ccmB	138077	138697	621	58.94%	41.06%	+	ATG	TGA		206aa
ccmC	374501	375316	816	55.76%	44.24%	+	ATG	TAG		271aa
ccmFC	288701	289455	1314	54.41%	45.59%	−	ATG	CGA		437aa
	287109	287667
ccmFN1	87565	88611	1047	54.25%	45.75%	+	ATG	TAA		348aa
ccmFN2	72574	73182	609	50.90%	49.10%	+	ATG	TGA		202aa
cob	132065	133255	1191	57.85%	42.15%	−	ATG	TGA		396aa
cox1	15812	17389	1578	56.97%	43.03%	−	GTG	TAA		525aa
cox2	99750	100143	789	58.05%	41.95%	+	ATG	TAA		262aa
	143966	144283
	145567	145643
cox3	90211	90909	699	56.51%	43.49%	+	ATG	TGA		232aa
matR	284822	286822	2001	49.03%	50.97%	−	ATG	TAG		666aa
mttB	39797	40546	750	55.33%	44.67%	+	ATG	TGA		249aa
nad1	123405	123789	978	57.06%	42.94%	+	ACG	TAA		325aa
	352743	352825
	351067	351258
	194608	194666
	283998	284256
nad2	218026	218178	1467	59.24%	40.76%	+	ATG	TAA		488aa
	219697	220088
	173248	173408
	171486	172058
	169816	170003
nad3	157436	157792	357	57.14%	42.86%	+	ATG	TAA		118aa
nad4	306216	306676	1488	58.20%	41.80%	−	ATG	TGA		495aa
	304319	304833
	300173	300595
	297678	297766
nad4L	224251	224553	303	64.03%	35.97%	−	ACG	TAA		100aa
nad5	265084	265313	2013	58.32%	41.68%	+	ATG	TAA		670aa
	266162	267377
	381969	381990
	68784	69178
	70318	70467
nad6	186485	187453	969	55.31%	44.69%	+	ATG	TAG		322aa
nad7	166802	166944	1185	55.53%	44.47%	−	ATG	TAG		394aa
	165414	165482
	163797	164263
	162309	162552
	160072	160333
nad9	325365	325862	498	57.23%	42.77%	+	ATG	TGA		165aa
rps12	157838	158215	378	55.03%	44.97%	+	ATG	TGA		125aa
rps12	274278	274520	243	58.02%	41.98%	+	ACT	TAG		80aa
trnM	157290	157356	67	46.27%	53.73%	+			CAU
trnE	17466	17537	72	50.00%	50.00%	−			UUC
trnK	42923	42995	73	50.68%	49.32%	−			UUU
trnL	278698	278778	81	50.62%	49.38%	+			CAA
trnfM	30450	30523	74	45.95%	54.05%	+			CAU
trnI	327445	327518	74	55.41%	44.59%	−			CAU
trnM	330298	330370	73	60.27%	39.73%	−			CAU
trnfM	380793	380866	74	43.24%	56.76%	+			CAU
trnQ	207503	207574	72	51.39%	48.61%	+			UUG
trnV	272758	272829	72	51.39%	48.61%	−			GAC
trnW	20333	20406	74	48.65%	51.35%	+			CCA
trnY	173964	174046	83	49.40%	50.60%	−			GUA
trnA	269635	269671	73	43.84%	56.16%	−			UGC
trnI	270710	270741	72	38.89%	61.11%	−			GAU

Discussion and conclusion

There are over 900 species in the genus Allium (Yang et al. 2023). Given the huge genome, molecular biology research on A. fistulosum has progressed slowly compared to that on other crops. Liao et al. (2022) published the genome of A. fistulosum, with a size of 11.27 Gb and pointed that A. fistulosum was the closest relative to A. cepa. In the present study, we assembled and analyzed the complete mitochondrial genome of A. fistulosum and found that A. fistulosum and A. cepa have a close genetic relationship. Not only that, the PCR identification result showed that cox2 gene in mitochondrial genome of A. fistulosum was trans-splicing, which further proof that A. fistulosum are closely related to A. cepa (Kim et al. 2013). The complete mitochondrial genome of A. fistulosum was reported for the first time. Our study lays the foundation for the study of the mitochondrial genome of the Allium genus and contributes to the identification and genetic, evolutionary, and functional studies of Amaryllidaceae species.

Author contributions

Jiayi Xing and Yongqin Wang designed the experiments, analyzed the data and wrote the manuscript. Mingzhao Zhu and Huiying Liu revised the manuscript and approved the version to be published. All authors agree to be accountable for all aspects of the work.

Ethical statement

The research and collection of plant material was carried out according to the guidelines of the Beijing Vegetable Research Center, Beijing Academy of Agricultural and Forestry Sciences. This study includes no endangered plant samples, and the sampling site is not located in any protected area.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The results of this study are authentic and effective, and the genome sequence data are publicly available in NCBI (https://www.ncbi.nlm.nih.gov/) under the accession number OL347690. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA782797, SRR17015068, and SAMN23401061, respectively.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China under Grant Number [31972409] and Science Innovation of the Beijing Academy of Agriculture and Forestry Sciences under Grant Number [KJCX20230123].