Ann. For. Res. 63(1): 3-18, 2020 ANNALS OF FOREST RESEARCH DOI: 10.15287/afr.2019.1413 www.afrjournal.org Structural, evolutionary and phylogenomic features of the plastid genome of Carya illinoinensis cv. Imperial Jordana Caroline Nagel1,2, Lilian de Oliveira Machado3, Rafael Plá Matielo Lemos1, Cristiane Barbosa D’Oliveira Matielo1, Tales Poletto4, Igor Poletto1, Valdir Marcos Stefenon3,1 Nagel J.C., de Oliveira Machado, L., Lemos R.P.M., Barbosa D’Oliveira Matielo C., Poletto T., Poletto I., Stefenon V.M., 2020. Structural, evolutionary and phylog- enomic features of the plastid genome of Carya illinoinensis cv. Imperial. Ann. For. Res. 63(1): 3-18. Abstract. The economically most important nut tree species in the world belong to family Juglandaceae, tribe Jungladeae. Evolutive investigations concerning spe- cies from this tribe are important for understanding the molecular basis driving the evolution and systematics of these species. In this study, we release the complete plastid genome of C. illinoinensis cv. Imperial. Using an IonTorrent NGS platform we generated 8.5 x 108 bp of raw sequences, enabling the assemblage of the com- plete plastid genome of this species. The plastid genome is 160,818 bp long, having a quadripartite structure with an LSC of 90,041bp, an SSC of 18,791 bp and twoIRs of 25,993 bp. A total of 78 protein-coding, 37 tRNA-coding, and 8 rRNA-coding regions were predicted. Bias in synonymous codon usage was detected in cultivar Imperial and three tRNA-coding regions were identified as hotspots of nucleotide divergence, with high estimations of dN/dS ratio. The high fraction of SSR loci prospected in non-coding regions may provide informative genetic markers, useful to a wide range of genetic researches. Despite the significant structural differences among plastid genomes, the phylogenetic relationships among species is supported by the whole plastid genome analysis, supporting the monophyly of subtribes Cary- inae and Juglandinae within family Juglandaceae. Keywords: Pecan, chloroplast genome, phylogenomics, Juglandaceae Authors. 1Federal University of the Pampa, Graduate Program in Biological Sciences, Campus São Gabriel, São Gabriel, RS, Brazil | 2Universidade Regional Integrada do Alto Uruguai e das Missões, Campus Santo Ângelo, Santo Ângelo, RS, Brazil | 3Federal University of Santa Catarina, Graduate Program in Plant Ge- netic Resources, Florianópolis, SC, Brazil | 4Federal University of Santa Maria, Graduate Program in Forest Engineering; Santa Maria, RS, Brazil. § Corresponding author: Valdir Marcos Stefenon ([email protected]) Manuscript received November 3, 2019; revised February 10, 2020; accepted Feb- ruary 18, 2020; online first March nd2 , 2020. 3 Ann. For. Res. 63(1): 3-18, 2020 Research article Introduction 2017, Lopes et al. 2017). Carya illinoinensis (Wangenh) K. Koch, In most plant species, the plastid DNA shows popularly known as pecan (Figure 1a-c, Sup- maternal inheritance, low recombination fre- porting Information), is a deciduous tree spe- quency, and a relaxed evolution rate. These cies of the tribe Junglandeae (Juglandaceae characteristics make plastid genomes substan- family), native to the temperate zones of North tially distinctive from the nuclear genomes America. Several species of tribe Juglandeae (Wolfe et al. 1987) and very useful in a wide - as Juglans regia (walnut), Cyclocarya pal- range of studies about evolutionary relation- liurus (wheel wingnut), Carya sinensis (Chi- ships in plants. The principal function of the nese hickory) and Carya illinoinensis (hickory plastid is to carry out photosynthesis, but oth- or pecan) - are important crop tree species cul- er major cellular functions also occur in this tivated in several regions of the world aiming organelle, including synthesis of starch, fatty at the commercial production of nuts. Pecan acids, pigments and amino acids (Wicke et al. is one of the most important nut crop species 2011). in the world, cultivated in several countries The known plastid genomes of green plants in North America (USA and Mexico), South typically contain from 100 to 120 genes, of America (Peru, Argentina, and Brazil), Afri- which approximately 70-88 are protein-coding ca (South Africa and Egypt), Asia (Israel and genes, 33-35 are tRNAs and four are rRNAs China) and Australia (Poletto et al. 2018). The (Wicke et al. 2011). With the progress of the species was introduced in Brazil with com- next generation sequencing (NGS) technolo- mercial interest during the 1870s and farmers gies, complete plastid genomes have been se- needed to select cultivars adapted to the cli- quenced for several plant species, generating a matic and biological conditions where the or- wide set of genomic resources, which enable chards were established (Poletto et al. 2015). the development of molecular markers and Currently, more than 40 cultivars are commer- more refined taxonomic and evolutionary stud- cially planted in large areas of Southern Bra- ies. The development of genetic markers from zil.Despite the economic significance of Carya plastid genome sequences has significantly as a crop tree species and the importance of contributed to studies about gene flow (plastid plastid genomesfor comparative evolutionary SSR markers; Perdereau et al. 2014), phylo- analysis and molecular taxonomy, only the geography (SNPs and Indels in plastid genes; plastid genomes of Carya sinensis (Hu et al. Stefenon et al. 2019a), and hybridization/in- 2016), Carya kweichowensis (Yeh et al. 2018), trogression (plastid SSR markers; Curtu et al. and Carya cathayensis (Zhai et al. 2019) were 2007) in forest tree species. Moreover, identifi- published to date, while one unpublished se- cation of genome rearrangements becomes in- quence of the plastid genome of Carya illi- creasingly important, especially as high levels noinensis is deposited in the NCBI database of rearrangement have been observed among (Genbank ID MH909599.1). Besides, few mo- both eukaryotes and prokaryotes (Wicke et al. lecular studies have been performed concern- 2011). Such rearrangements may also be very ing C. illinoinensis cultivars planted in south- useful to understand phylogenetic and evolu- ern Brazil (e.g Poletto et al. 2019). Considering tionary trends within and among plant groups. the scarcity of genomic studies available for Knowledge about genome rearrangements, Carya species and aiming to generate novel gene content, recombination events, loss of genomic resources for Carya illinoinensis, we genes, and gene transfer to the nucleus are of sequenced, assembled and characterized the great importance for understanding evolution- complete plastid genome of C. illinoinensis ary events in plants (Vieira et al. 2016, Bock cv. Imperial using next-generation sequencing 4 Nagel et al. Structural, evolutionary and phylogenomic features ... (NGS) technology. Here, we report the main mined as the total size of the sequenced reads finds obtained from this initiative concerning divided by the size of the assembled plastid the genes present and the codon usage bias in genome.Annotation of the plastid genome was the plastid genome of this species. In addition, conducted using the GeSeq (Tillich et al 2017) we identified potentially polymorphic plastid and the cpGAVAS (Liu et al. 2012) platforms. SSR markers and also performed a compara- For GeSeq, annotation started from four ref- tive evolutionary analysis of plastid genomes erences chloroplast annotations (Arabidopsis between C. illinoinensis cv. Imperial and other thaliana, Castanea mollissima, Juglans nigra tree species of the tribe Juglandeae using pub- and J. regia). The software tRNAscan (Chan licly available genomic datasets, thus reveal- and Lowe 2019) and Aragorn (Laslett and ing some features of pecan evolution. Camback 2004) were used for searching the tRNAs, while the physical circular map of the plastid genome was built using Organellar Ge- Materials and methods nome DRAW software (Lohse et al. 2013). Plastid genome sequencing and assembling Characterization of plastid genome features in C. illinoinensis cv. Imperial Healthy leaves of an adult individual of C. illinoinensis cv. Imperial were sampled in Relative synonymous codon usage (RSCU) of the municipality of Anta Gorda, Rio Grande all protein-coding genes were determined us- do Sul State, southern Brazil. A voucher of ing MEGA 6.0 software (Tamura et al. 2013). this sample was deposited under the number RSCU corresponds to the proportion of the ob- HBEI1624 in the Bruno Edgar Irgang Herbar- served occurrence of a codon to its expected ium of the Federal University of the Pampa, occurrence if all the synonymous codons of a Brazil. Intact chloroplasts were isolated from particular amino acid are used evenly. the leaves as described by Matielo et al. (2019) Prospection of simple sequence repeats (SSRs) and the plastid DNA (cpDNA) was isolated us- loci in the plastid genome of C. illinoinensis cv. ing the CTAB method (Doyle and Doyle 1987). Imperial was performed using the Perl script The quality of the isolated DNA was checked MISA (Beier et al. 2017) setting minimum using a NanoVueTM spectrophotometer (GE thresholds for search at ten for mononucleo- Healthcare). The isolated cpDNA was used tide repeats, six for dinucleotide repeats and for library preparation with Ion OneTouch2™ five for tri-, tetra-, penta- and hexanucleotide System using the Ion PGM™ Template OT2 repeats. The location of the SSR loci within 400 Kit. The sequencing was performed us- the plastid genome was determined using