Characterization of the complete plastid genome of Butia eriospatha (Arecaceae)

Abstract

Butia eriospatha is an endemic palm species from the Atlantic Rainforest in Brazil, a biodiversity hotspot. This species is currently listed in the IUCN red list as vulnerable and lacks specific plastid markers for population genetics studies. In addition, the evolutionary relationship within the genus Butia is not yet well resolved. Here, we sequenced and characterized the complete plastid genome (plastome) sequence of B. eriospatha. The complete plastome sequence is 154,048 bp in length, with the typical quadripartite structure. This plastome length and genes content is consistent with other six species from tribe Cocoseae. However, the Inverted Repeat (IR) borders show some variation among the analyzed species from this tribe. Species from the Bactridinae (Astrocaryum and Acrocomia) and Elaeidinae (Elaeis) subtribes present the rps19 gene completely duplicated in the IR region. In contrast, all plastomes sequenced from the subtribe Attaleinae (Butia, Cocos, Syagrus) present one complete CDS of rps19 and one partial copy of rps19. The difference in the IR/LSC junctions between Attaleinae and the sister clades Bactridinae + Elaeidinae might be considered an evolutionary signal and the plastome sequence of B. eriopatha may be used in future studies of population genetics and phylogeny.

The family Arecaceae, comprises approximately 188 genera and 2,585 species, distributed throughout tropical and subtropical climate of the world, but it is most diverse in tropical forest habitats, being present from Americas to Asia-Pacific region (Palmweb).

Butia (Becc.) Becc. is a genus of the family Arecaceae, indigenous from South America. This genus includes 18 species (WFO, 2019), among them Butia eriospatha (Mart. ex Drude) Becc.. This species is endemic to southern Brazil and occurs in the Atlantic Rainforest phytogeographic domain: mainly in high altitude grassland, grassland, palm grove (Heiden et al., 2020). Butia eriospatha is easily distinguished by its solitary stem with 4-5 meters in hight, globose fruit, spathe ferruginous-pubescent on outer surface, and pistillate flowers with 3-7 mm (Deble et al., 2011; Heiden et al., 2020). Among palms, B. eriospatha has a high economic importance, being used for ornamentals projects, and with edible fruits. Currently, the fruits are consumed fresh or in pulp, alcoholic drinks, jams, jellies or ice cream (Hoffmann et al., 2014).

Species of the genus Butia show the same chromosome number (2n = 2x = 32) (Correa et al., 2019) and the genus is well supported as monophyletic (Meerow et al., 2015). However, within Butia, few molecular markers were used in phylogenetic studies. The most recent study on Butia phylogeny used only one plastid marker (trnH-psbA) and two nuclear markers (ITS and WRKY19) and failed to resolve the evolutionary relationship between Butia species (Pereira, 2019).

Plastome sequences are frequently used to understand evolutionary events and efficiently resolve phylogenetic relationships (Lopes et al., 2018, 2019). Typically, the plastome is represented as a single circular molecule composed by two inverted regions (IR), a large single copy (LSC) and a small single copy (SSC), with 120 to 130 genes, encoding ribosomal RNA (rRNA), transfer RNA (tRNA) and peptides. The plastome size varies from 107 kb to 218 kb, depending on each species (Daniell et al., 2016; Menezes et al., 2018).

Here we report the complete plastome of B. eriospatha and describe its structure and gene composition. For this, B. eriospatha leaves were collected from a single individual at the Federal University of Santa Catarina - Brazil. DNA isolation with plastid DNA-enrichment was performed according to Vieira et al. (2014). The DNA was quantified using Qubit^® fluorometer (Invitrogen, Carlsbad, CA) and 1 ng of DNA was used for preparing sequencing libraries with Nextera XT DNA Sample Prep Kit (Illumina Inc., San Diego, CA). Libraries were sequenced on Illumina MiSeq (Illumina Inc.). The obtained paired-end reads (2 x 300 bp) were used for de novo assembly in CLC Genomics Workbench 8.0v. The plastome annotation was performed using DOGMA software (Wyman et al., 2004) followed by manual curation using Geneious (Kearse et al., 2012). The graphical map of plastomes was generated by OrganellarGenomeDRAW (OGDRAW) (Greiner et al., 2019). The complete nucleotide sequence of B. eriospatha (MN329806) plastome was deposited in the GenBank database. The junction site of plastomes (LSC/IR, IR/SSC) from the tribe Cocoseae (Arecaceae: Arecoideae) were visualized and compared using IRScope (Amiryousefi et al., 2018).

Butia eriospatha plastome is a circular molecule with 154,048 bp in length and the typical quadripartite structure, including a large single copy with 83,805 bp, two inverted repeat regions (26,437 bp) and a small single copy with 17,369 bp (Figure 1). This plastome assembly was realized with 457,423 plastid reads (all trimmed with 0.05 quality scores in CLC Genomics Workbench), which resulted in approximately 820x genome coverage depth. The minimum genome coverage was 206x, which shows the high coverage sequencing of this plastome.

Figure 1. Graphical map of Butia eriospatha plastid genome. Genes shown on the outside of the circle are transcribed clockwise; genes on the inside are transcribed counterclockwise. Genes were grouped and represented by different colors according their function group. The GC content is represented by the dashed darker grey area in the inner circle, the lighter grey area represents AT content.

This plastome encodes 113 unique genes, being 79 protein-code genes, 30 tRNA genes and four rRNA genes (Table 1), the same number as the other Cocoseae species (Lopes et al., 2018, 2019). The 20 duplicated genes in the IRs are: eight protein-coding genes (among them, the ycf1 and rps19 are partially duplicated), eight tRNA genes, and all four rRNA genes. Among the 113 genes, 16 contain one intron (10 protein-codes genes, and six rRNA), and three contain two introns (clpP, ycf3 and rps12) (Table 1).

Table 1. List of genes identified in the plastome of Butia eriospatha.

Group of genes	Name of gene
Gene expression machinery
Ribossomal RNA genes	rrn4.5 a ; rrn5 a ; rrn16 a ; rrn23 a
Transfer RNA genes	trnA-UGC ab ; trnC-GCA; trnD-GUC; trnE-UUC; trnF-GAA; trnfM-CAU; trnG-UCC b ; trnG-GCC; trnH-GUG a ; trnI-CAU a ; trnI-GAU a b ; trnK-UUU b ; trnL-CAA a ; trnL-UAA b ; trnL-UAG; trnM-CAU; trnN-GUU a ; trnP-UGG; trnQ-UUG; trnR-ACG a ; trnR-UCU; trnS-GCU; trnS-UGA; trnS-GGA; trnT-UGU; trnT-GGU; trnV-GAC a ; trnV-UAC b ; trnW-CCA; trnY-GUA
Small subunit of Ribossome	rps2; rps3; rps4; rps7 a ; rps8; rps11; rps12 ab ; rps14; rps15; rps16 b ; rps18; rps19 a
Large subunit of Ribossome	rpl2 ab ; rpl4; rpl16 b ; rpl20; rpl22; rpl23 a ; rpl32; rpl33; rpl36
DNA-dependent RNA Polymerase	rpoA; rpoB; rpoC1 b ; rpoC2
Translational initiation factor	infA
Maturase	matK
Genes for photosynthesis
Subunits of photosystem I (PSI)	psaA; psaB; psaC; psaI; psaJ; ycf3 b ; ycf4
Subunits of photosystem II (PSII)	psbA; psbB; psbC; psbD; psbE; psbF; psbH; psbI; psbJ; psbK; psbL psbM; psbN; psbT; psbZ
Subunits of cytochrome b6f	petA; petB b ; petD b ; petG; petL; petN
Subunits of ATP synthase	atpA; atpB; atpE; atpF b ; atpH; atpI
Subunits of NADH dehydrogenase	ndhA b ; ndhB ab ; ndhC; ndhD; ndhE; ndhF; ndhG; ndhH; ndhI; ndhJ; ndhK
Large subunit of Rubisco	rbcL
Other functions
Envelope membrane protein	cemA
Subunit of acetyl-CoA carboxylase	accD
C-type cytochrome synthesis	ccsA
Subunit of protease Clp	clpP b
Component of TIC complex	ycf1
Unknown function	ycf2 a

^a Duplicated genes;

^b Genes containing introns

The size of the plastome of B. eriospatha was compared with others plastomes from species in the same tribe: Cocoseae (Table 2). They have similar length and structure, varying in the LSC from 83,805 bp to 85,192 bp, the SSC from 17,369 bp to 17,639 bp and the IRs from 26,437 bp to 27,092 bp. This variation can be better visualized in the IRs borders (Figure 2). Species from the subtribes Bactridinae (Acrocomia and Astrocaryum) and Elaeidinae (Elaeis) present the rps19 gene completely duplicated in the IR region. In contrast, all plastomes sequenced from subtribe Attaleinae (Butia, Cocos, Syagrus) present one complete CDS of rps19 and one partial copy of rps19, thus presenting only one functional copy of the gene (Figure 2). The intergenic spacers (IGS) between rpl22-rps19 genes and rps19-psbA genes, located in the LSC/IR_B and IR_A/LSC junctions, respectively, show variations in size within Cocoseae species (Figure 2). The SSC/IR_A junction varies mainly in the length of the ycf1 gene portion contained within the SSC. The ndhF gene is identical in length among all species studied and part of it is located within the IR_B, overlapping in part the ycf1 gene (56 bp). The differences in the IR/LSC junctions between Attaleinae and the sister clades Bactridinae + Elaeidinae migth be considered an evolutionary signal and the plastome sequence of B. eriospatha may be used in future studies of population genetics and for phylogenetic studies.

Table 2. General features of plastid genomes within the tribe Cocoseae.

Subtribe	Species	Total (bp)	LSC (bp)	IR (bp)	SSC (bp)	Accession number
Attaleinae	Butia eriospatha	154,048	83,805	26,437	17,369	MN_329806
	Syagrus coronata	155,053	84,535	26,522	17,474	NC_029241
	Cocos nucifera	154,731	84,230	26,555	17,391	NC_022417
Elaeidinae	Elaeis guineensis	156,973	85,192	27,071	17,639	NC_017602
Bactridinae	Astrocaryum aculeatum	156,804	85,037	27,081	17,605	MH_537788
	Astrocaryum murumuru	156,801	85,017	27,081	17,622	MH_537787
	Acrocomia aculeata	156,500	84,936	27,092	17,380	NC_037084

Figure 2. Comparison of plastid genome LSC, IR, and SSC junction positions within the tribe Cocoseae (Arecaceae: Arecoideae). Genes shown below are transcribed counterclockwise and those shown above the lines are transcribed clockwise.

Conflict of Interest

The authors declare that they do not have any conflict of interest.

Author Contributions

LNV, MPG, EMS, MR, and HPFF conceptualized the experiment; JWSM, LNV, HPFF and RSS performed the experiments and data analysis; EMS, MR, and MPG provided the reagents and materials; JWSM, LNV, and HPFF wrote the manuscript. All authors revised the final version of the manuscript.