Complete chloroplast genome sequence and phylogenetic analysis of Annona reticulata

Abstract Annona reticulata is native to South and Central America which has many phytochemical and pharmacological activities suggesting a wide range of clinical application in lieu of cancer chemotherapy. This study provides abundant genomic data for the genetic relationship study, germplasm resources evaluation and varieties selection of A. reticulata. The complete chloroplast genome of A. reticulata was sequenced, assembled, and annotated in this study. The genome size was 201,906 bp and was divided into four regions: a large single-copy region of 69,650 bp, a small single-copy region of 3,014 bp, and two inverted repeat regions of 64,621 bp. A total number of 164 genes were annotated, including 115 protein-coding genes, one pseudogene, 40 tRNA genes, and eight rRNA genes. In terms of gene function, the 164 genes were divided into four major groups: genes for self-replication, photosynthesis, unknown function, and other genes. A maximum likelihood tree based on the chloroplast genome sequences of 24 plant species was constructed. The result of phylogenetic analysis showed that A. cherimola had the closest relationship with A. reticulate.

Annona reticulata is a medium sized plant, native to South and Central America, belonging to the Annonaceae family (Mondal et al. 2008). The Annona genus consists of about 119 species, most of which are shrubs and trees, and are widely distributed in the tropical and subtropical regions (Thang et al. 2013). The fruit of A. reticulata has a smooth skin, which becomes dull red when ripe. It is commonly known as bullock's heart or custard apple. Its custard like pulp is rather sugary and less flavored as compared to other familiar species, so it's usually eaten as a dessert fruit and made into drinks and ice creams (Jorge et al. 2003;Ogunwande et al. 2006). Besides, A. reticulate has been used as a traditional medicine in some of the southeast Asian countries (Mondal et al. 2007), such as India, Malaysia, Indonesia, Thailand, Vietnam and some other countries. The phytochemical and pharmacological activities of A. reticulata components suggest a wide range of clinical applications in lieu of cancer chemotherapy (Sureshet al. 2011), and it is having various other pharmacological activities, such as antioxidant, analgesic and CNS depressant, antimalarial, anthelmintic, syphilis, and few more (Chavan 2014).
In the recent times, many studies have focused on the isolation, characterization and utilization of natural antioxidants of A. reticulata (Suneelkumar et al. 2011). However, there has been little research on its genome. The chloroplast genome is a relatively independent genetic system in the plant cells that contains abundant genetic information, which encodes many genes relating to photosynthesis and other important biological processes. Research on chloroplast genome is very important for analysis of plant evolution, genetic relationship identification and germplasm resource evaluation. In this study, the complete chloroplast genome of A. reticulata was sequenced, assembled, and annotated.
The specimen of A. reticulata was collected from from the Xishuangbanna Tropical Flowers and Plants Garden (100.70422 E, 22.015885 N) and deposited in the herbarium of Yunnan Institute of Tropical Crops (Xishuangbanna, China) with the specimen voucher number of YITC-2020-FZ-A-004. The genomic DNA extraction was done by using the Dneasy Plant Mini Kit (Qiagen). DNA sample quality and quantity were characterized by gel electrophoresis and Nano-Drop 2000 spectrometer (Thermo Fisher Scientific, USA). The high-quality genomic DNA were used to prepare DNA library following the manufacturer's instructions (Illumina, San Diego, CA) with insert sizes of 350 bp for paired-end sequencing, paired-end (PE) sequencing was conducted on the Illumina Hiseq 2500 Platform (Illumina, San Diego, CA). The chloroplast genome of A. reticulata was assembled by CLC Genomics Workbench v3.6 (http://www.clcbio.com) and annotated by DOGMA (Wyman et al. 2004), using the cp annotation of Annona cherimola (NC_030166) as a reference, and then uploaded to the GenBank (http://www.ncbi.nlm.nih.gov/) with the accession number MT742547.
A maximum likelihood tree based on the chloroplast genome sequences of 24 plant species was constructed to study the phylogenetic relationship between A. reticulata and other plant species (Figure 1). The jModelTest 2.1.7 (David 2008) software was employed to analyze nucleotide substitutions model under the Akaike Information Criterion (AIC), the GTR þ G þ I model was selected for nucleotide and the phylogenetic analysis was carried out using the maximum likelihood method with the RAxML8.1 (Alexandros 2006). Statistical supports were assessed with 1000 bootstrap replicates. Out of 24 plant species, 17 species belonged to Magnoliaceae family, five to Annonaceae family, one to Myristicaceae family and Trochodendron aralioides belonged to Trochodendraceae family, which was used as the out group. Multiple sequence alignment was carried out by MAFFT (Katoh and Standley 2013) and maximum-likelihood (ML) analysis was carried out by MEGA7.0 (Kumar et al. 2016). Phylogenetic analysis indicated that Annona cherimola had the closest relationship with A. reticulata. This study provides abundant genomic data for the genetic relationship study, germplasm resources evaluation and varieties selection of A. reticulata.

Disclosure statement
The authors report no conflict of interest.

Data availability statement
The chloroplast genome sequence data that support the findings of this study are openly available in GenBank at https://www.ncbi.nlm.nih.gov/, reference number MT742547.
The raw sequencing data are openly available in SRA database with the accession number PRJNA658442 and SRR12506405.