The Complete Chloroplast Genome of “Lasona” (Allium cepa var. Aggregatum G. Don), an Important Indigenous Vegetable in the Northern Philippines

Allium cepa var. aggregatum Don (1827) is locally referred to as “lasona,” an important culinary spice and vegetable, particularly in the Northern parts of the Philippines. It is a field-grown bulb belonging to the family of Amaryllidaceae. Otherwise known as shallots, potato onion, or multiplier onion, it is a distinct form in the aggregatum group placed under one of the horticultural classifications of A. cepa L. (Jones and Mann, 1963). It is characterized by having round, depressed, aggregated bulbs with brown outer coats and undeveloped scape (Don, 1827; Jiemei and Kamelin, 2000). The genus Allium, which includes economically significant bulbous vegetables such as onion, is considered native to southwestern Asia. However, it is now cultivated globally (Devi et al., 2015; Rahaman et al., 2021). In general, the center of origin of the genus Allium extensively spreads from the Mediterranean basin to central Asia (van der Meer et al., 1997).
               
A. cepa var. aggregatum largely contributes to the human diet and is being utilized as an ornamental, natural alternative to synthetic food preservatives and for medicinal purposes (Saraswathi et al., 2017; Shabir et al., 2020; van der Meer et al., 1997). In the Philippines, it is commonly grown for greens and cooked as a vegetable called “lasona gulay”. Along with yellow and red bulbs, “lasona” is one of the most popular varieties of onions, alongside garlic, traded in the country (DA-BAR, 2021). Despite the numerous and distinct variations in appearance, flavor and usage, the differentiation between Allium cultivars-particularly onions-remains challenging. This difficulty arises due to the complex nature of Allium species and their hybrids, which often exhibit overlapping characteristics (Shabir et al., 2020). Nevertheless, considering these vegetables’ widespread cultivation and economic significance, a comprehensive understanding of their genetic variability is indispensable (Rahaman et al., 2021; Ananthan and Balakrishnamoorthy). Hence, we assembled, characterized and compared the chloroplast genome of “lasona” (A. cepa var. aggregatum) with other A. cepa accessions. The assembled plastome can be utilized as a resource for distinguishing the taxon and elucidating its evolutionary relationship with other Allium species, particularly within the aggregatum group.

Characterization of plant material
 
 A. cepa var. aggregatum were collected from the field gene bank of the Crop Breeding and Genetic Resources Division, Institute of Crop Science University of the Philippines Los Baños, Laguna. Its type locality is at San Nicolas, Ilocos Norte (18^o7' 59.7105", 120^o36' 10.71"). The prepared voucher specimen (ICROPS 1374) was deposited in the Philippine Herbarium of Cultivated Plants of the Institute of Crop Science, University of the Philippines Los Baños, Laguna, Philippines (https://cafs.uplb.edu.ph/icrops/, Dr. Renerio P. Gentallan Jr., rpgentallan@up.edu.ph. Subsequently, the morphology of the accession was characterized based on the prominent morphological markers in the Allium cepa group described by Don (1827) to verify the identity of the utilized accession. The whole plant, inflorescence and fruits were photographed in situ and ex situ. The quantitative traits measured are reported as the mean±standard deviation of the mean while qualitative traits were observed.

DNA extraction, chloroplast genome sequencing, assembly and annotation
 
Fresh A. cepa var. aggregatum leaves (0.5 g) were used to extract genomic DNA using the slightly modified CTAB method by Doyle and Doyle (1987). Sequencing was performed using the HiSeq-PE150 platform (Illumina Inc., San Diego, CA, USA) by NovogeneAIT Genomics Singapore PTE LTD (Singapore).  The 150-bp paired-end raw reads (20278894) were produced which were subsequently filtered to generate 20248894 of cleaned reads. Using GetOrganelle v1.7.5+ software (Jin et al., 2020), these were assembled into a circular genome. The circularized genome was annotated, then mapped using CPGAVAS2 (Shi et al., 2019) and subsequently visualized using OGDRAW (Greiner et al., 2019). The assembled cp genome sequence was submitted to GenBank with the accession number OP756522 of the National Center for Biotechnology Information (NCBI).
 
Phylogenetic analysis and genome comparison
 
The assembled chloroplast genome sequence of Allium cepa var. aggregatum, along with the complete chloroplast genome sequences of 33 Amaryllidaceae species available from the NCBI website, were used to elucidate phylogenetic relationships. The multiple sequence alignment was performed using MAFFT (Katoh and Standley 2013) and a Maximum Likelihood (ML) phylogenetic tree was constructed using the Hasegawa-Kishino-Yano, Gamma distribution (HKY+G) model (Hasegawa et al., 1985) with 1,000 bootstrap replicates in MEGA-XI (Tamura et al., 2021). To determine and visualize the contraction and expansion of IR regions, the chloroplast genome of Allium cepa var. aggregatum was compared with other closely related Allium species using IRScope (Amiryousefi et al., 2018).

Morphological characterization of the plant material
 
The morphological characteristics of the accession (ICROPS 1374) align with the descriptions provided by Don (1827). Allium cepa var. aggregatum (ICROPS 1374; Fig 1) exhibits green foliage with a shaft that is 29.67±1.34 mm long and 3.52±0.23 mm in diameter. The bulb is broadly elliptic, aggregated, with thin deep purplish red skin and white flesh. The bulbs also have protective bulb-coat leaves, purplish, brown, or white (Geerinck, 1993). Each aggregate is composed of 5 to 8 bulbs, this “dividing” habit constantly appears in breeding lines of the common large-bulbed forms of A. cepa (Jones and Mann, 1963).



Complete genome assembly and annotation of Allium cepa var. aggregatum
 
The assembled complete cp genome of Allium cepa var. aggregatum (GenBank no. OP756522) had a typical quadripartite circular structure and a sequence length of 153,587 bp, which is longer than the chloroplast genomes of its closest related Allium species, Allium cepa (153,529 bp; KM088013.1), Allium cepa strain CMS-T (153,568 bp; KM088015.1) and Allium cepa genotype normal (N) (153,538 bp; KF728080.1) available in NCBI. It includes a pair of inverted repeats (IRa and IRb) with 26,468 bp each that separates the rest of the genome sequences into two single-copy regions:  LSC region (17,931 bp in length) and SSC region (17,031 bp in length) (Fig 2). The overall GC content of the cp genome is 36.8% with base compositions of 31.3% A, 31.9% T, 18.1% G and 18.7% C. A total of 126 coding genes were annotated in the cp genome of A. cepa var aggregatum comprising 38 tRNA genes, 8 rRNA genes and 80 protein-coding genes. Based on the functions of these genes, there are 43 photosynthesis-related genes, 29 self-replication genes, 6 other genes with different functions (matK, cemA, accD, clpP, cCsA and inFa) and 1 unknown conserved open reading frame (Table 1). This is the first reported complete chloroplast genome of A. cepa var. aggregatum.





Phylogenetic analysis
 
Phylogenetic analysis showed that A. cepa var. aggregatum clustered with other Allium cepa accessions to form a distinct clade. However, despite their close relationship, A. cepa var. aggregatum was markedly separated from the six other A. cepa accessions with strong bootstrap support (Fig 3). The observed topology within the A. cepa clade may be attributed to infraspecific differences initially observed in this taxon with 19 recorded botanical varieties that are presently considered as heterotypic synonyms (POWO, 2022). This indicates that infraspecific plastome differences are observed between A. cepa individuals, unlike other taxa with conserved plastome sequences across accessions (Waters and Schaal, 1991; Agrawal et al., 2014; Liu et al., 2021). Moreover, we observed that A. cepa accessions were closely related to A. schoenoprasum. A similar relationship, within and between species, was resolved by Jimenez et al., (2020) where they included plastomes of fertile and sterile strains of A. cepa in their phylogenetic analysis of Allium species in the Amaryllidaceae family.


 
Plastome comparison
 
Inverted Repeat (IR) lengths of Allium cp genomes ranged from 12,186 bp to 26,468 bp with A. cepa genotype male sterile (S) and A. cepa genotype normal (N) having the shortest and longest IR regions, respectively. Differences were observed across all junction sites among the seven A. cepa plastomes (Fig 4) rps19 and rpl22 were distributed in LSC/IRb boundary across six A. cepa accessions with varying distances of two genes to the junction. Only the rpl22 of A. cepa var. aggregatum was placed 30 bp away from the LSC/IRb boundary. For SSC/IRa boundaries, the ycf1 gene located on the SSC region had a varying extension length to the IRa regions between the six A. cepa accessions while this gene was absent in A. cepa var. aggregatum. The ndhF genes, located primarily in the SSC region, were extended by 1 bp and overlapped the IRb region for five out of seven A. cepa genotypes at the IRb/SSC boundary. Moreover, there is also differential placement of psbA gene in reference to the IRa/LSC junction. The size variations, as observed in the seven plastomes sequenced from different A. cepa, may be attributed to the expansion and contraction of the IR and SSC junction sites (Amiryousefi et al., 2018). The compositions of these genomes were also different in terms of the number of mRNA and rRNA genes (Table 2). These observed variations, along with the differences in the junction sites, provide additional resolution to the differences observed in the A. cepa plastomes. This further supports the distinctness of the A. cepa var. aggregatum plastome sequenced. These recorded chloroplast genome variations could further facilitate generation of DNA barcodes needed for germplasm identification at the variety level.

The complete chloroplast genome of an accession of A. cepa var. aggregatum was assembled and annotated and the morphology of the genotyped accession was also successfully characterized. The complete chloroplast (cp) genome is 153,587-bp long with 126 coding genes comprising 80 mRNA genes, 38 tRNA genes and 8 rRNA genes. Out of the mRNA genes, 43 genes code for photosynthesis, 29 genes for self-replication and 6 other genes for other functions. This is the first complete chloroplast genome of Allium cepa var. aggregatum. A phylogenetic analysis of the assembled genome, along with 21 other Allium accessions and 12 other Amaryllidaceae species, revealed that A. cepa var. aggregatum formed a distinct clade despite infraspecific plastome variations. The chloroplast genome was also distinct in sequence length and composition. This reported complete chloroplast genome of A. cepa var. aggregatum could be a valuable resource to further clarify differences needed for varietal identification of Allium species. 

The authors would also like to thank Ronil Beliber and Arvin Medrano for cultivating the germplasm and Eddelaine Joyce Bautista and Edna Mercado for processing the paperwork needed for the research project.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
There are no ethical issues associated with the plant material used in the study. The species is not endangered and not on the CITES and IUCN Red list and the sample was not collected from a protected area. The plant materials were collected from the field gene bank of the Crop Breeding and Genetic Resources Division, Institute of Crop Science, University of the Philippines Los Baños, Laguna, in compliance with the guidelines of the Institute of Crop Science.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.