diversity Article Complete Chloroplast Genome Sequence and Comparative and Phylogenetic Analyses of the Cultivated Cyperus esculentus Wei Ren 1,†, Dongquan Guo 1,†, Guojie Xing 1,†, Chunming Yang 1, Yuanyu Zhang 1, Jing Yang 1, Lu Niu 1, Xiaofang Zhong 1, Qianqian Zhao 1, Yang Cui 1, Yongguo Zhao 2,* and Xiangdong Yang 1,* 1 Jilin Provincial Key Laboratory of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun 130024, China; [email protected] (W.R.); [email protected] (D.G.); [email protected] (G.X.); [email protected] (C.Y.); [email protected] (Y.Z.); [email protected] (J.Y.); [email protected] (L.N.); [email protected] (X.Z.); [email protected] (Q.Z.); [email protected] (Y.C.) 2 College of Biology and Food Engineering, Guangdong University of Petrochemical Technology, Maoming 525000, China * Correspondence: [email protected] (Y.Z.); [email protected] (X.Y.) † These authors contributed equally to this study. Abstract: Cyperus esculentus produces large amounts of oil as one of the main oil storage reserves in underground tubers, making this crop species not only a promising resource for edible oil and biofuel in food and chemical industry, but also a model system for studying oil accumulation in non-seed tissues. In this study, we determined the chloroplast genome sequence of the cultivated C. esculentus (var. sativus Boeckeler). The results showed that the complete chloroplast genome of C. esculentus was 186,255 bp in size, and possessed a typical quadripartite structure containing one large single copy (100,940 bp) region, one small single copy (10,439 bp) region, and a pair of Citation: Ren, W.; Guo, D.; Xing, G.; inverted repeat regions of 37,438 bp in size. Sequence analyses indicated that the chloroplast genome Yang, C.; Zhang, Y.; Yang, J.; Niu, L.; encodes 141 genes, including 93 protein-coding genes, 40 transfer RNA genes, and 8 ribosomal RNA Zhong, X.; Zhao, Q.; Cui, Y.; et al. genes. We also identified 396 simple-sequence repeats and 49 long repeats, including 15 forward Complete Chloroplast Genome repeats and 34 palindromes within the chloroplast genome of C. esculentus. Most of these repeats Sequence and Comparative and were distributed in the noncoding regions. Whole chloroplast genome comparison with those of the Phylogenetic Analyses of the Cultivated Cyperus esculentus. other four Cyperus species indicated that both the large single copy and inverted repeat regions were Diversity 2021, 13, 405. https:// more divergent than the small single copy region, with the highest variation found in the inverted doi.org/10.3390/d13090405 repeat regions. In the phylogenetic trees based on the complete chloroplast genomes of 13 species, all five Cyperus species within the Cyperaceae formed a clade, and C. esculentus was evolutionarily Academic Editor: Mario A. Pagnotta more related to C. rotundus than to the other three Cyperus species. In summary, the chloroplast genome sequence of the cultivated C. esculentus provides a valuable genomic resource for species Received: 1 July 2021 identification, evolution, and comparative genomic research on this crop species and other Cyperus Accepted: 22 August 2021 species in the Cyperaceae family. Published: 26 August 2021 Keywords: Cyperus esculentus; chloroplast genome; comparative analysis; phylogeny Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Cyperus esculentus L., also known as yellow tigernut, yellow nutsedge, or chufa, is a perennial C4 plant in the sedge family (Cyperaceae), which is comprised of approximately 5500 species worldwide. It occurs as wild or cultivated varieties and exhibits ecological Copyright: © 2021 by the authors. plasticity and a wide global distribution [1]. Cultivated C. esculentus (var. sativus Boeckeler) Licensee MDPI, Basel, Switzerland. originated in the Mediterranean area, where it has been grown for its edible tubers since This article is an open access article Cyperus esculentus distributed under the terms and pre-dynastic Egypt (fourth millennium BC) [1]. has been reported to conditions of the Creative Commons contain approximately 40% starch, 30% oil, 20% sugar, 9% protein, 6% fiber, and high levels Attribution (CC BY) license (https:// of vitamins E and C in its tubers [2]. In contrast to oil-bearing crops that mainly produce creativecommons.org/licenses/by/ oil in seeds as well as the mesocarp of certain fruits, C. esculentus might be the only plant 4.0/). known that accumulates a large amount of oil as one of the main storage reserves in its Diversity 2021, 13, 405. https://doi.org/10.3390/d13090405 https://www.mdpi.com/journal/diversity Diversity 2021, 13, 405 2 of 14 underground tubers [2]. The unique characteristics of the species make it a promising resource for producing edible oil and biofuel in the food and chemical industries, and also provide a novel model system for studying oil accumulation in non-seed tissues [3]. Additionally, as a medicine, C. esculentus has been reported to help boost blood circulation, reduce cardiovascular diseases and heart attacks, and prevent stroke and inflammation in the respiratory passages [4–8]. Previous studies revealed that C. esculentus can inhibit free radicals and/or key enzymes involved in starch digestion, such as α-amylase and α-glucosidase, making this plant a dietary control option for patients with diabetes [9]. As active metabolic centers responsible for photosynthesis and the synthesis of amino acids, nucleotides, fatty acids, phytohormones, vitamins, and other metabolites, chloro- plasts play important roles in the physiology and development of land plants and al- gae [10,11]. In most land plants, the chloroplast genomes exhibit highly conserved struc- tures and organization, and typically exist as circular DNA molecules with a size of 120–170 kb [12]. Chloroplast genomes generally have a quadripartite structure and contain a large single copy (LSC) region and small single-copy (SSC) region separated by inverted repeats (IRs), although these IR regions are missing in some species [13,14]. Specific charac- teristics of the chloroplast genome, such as its maternal inheritance, haploid nature, and low level of recombination, make it a robust tool for genomics and phylogenetic studies of several plant families [15–18]. Moreover, considerable variations within chloroplast genomes can also provide useful information for evaluating the phylogenetic relationships of taxonomically unresolved plant taxa and understanding the relationship between plant nuclear, chloroplast, and mitochondrial genomes in plants [17,19–21]. C. esculentus exhibits remarkable variability with several morphotypes because of its ecological plasticity and wide distribution. However, very little information on the nuclear, chloroplast, and mitochondrial genomes of C. esculentus has been reported. Here, we assembled and annotated the complete chloroplast genome of cultivated C. esculentus. Comparative analysis of the chloroplast genomes of Cyperus species was conducted, and the phylogenetic position of C. esculentus in the Cyperaceae family was inferred. 2. Materials and Methods 2.1. Plant Materials and Genomic DNA Extraction Cultivated C. esculentus was collected from plants grown in Wuhan, China in 2017, and voucher specimen (herbarium voucher No. JYD-2) was maintained at the Jilin Academy of Agricultural Sciences, Changchun, China. Fresh leaves of the plants were collected and immediately stored at −80 ◦C until analysis. Total genomic DNA was extracted using the modified CTAB method [22]. The integrity, quality, and concentration of the DNA were determined by agarose gel electrophoresis and a NanoDrop spectrophotometer 2000 (Thermo Fisher Scientific, Waltham, MA, USA). 2.2. DNA Sequencing and Genome Assembly High-quality genomic DNA was used to construct libraries with an average length of 350 bp using the NexteraXT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) and sequenced on the Illumina Noveseq 6000 platform (Illumina). More than 18.5 million paired-end reads with an average length of 150 bp were generated and edited using the NGS QC Tool Kit v2.3.3 [23]. Complete circular assembly graph was checked and further extracted by visualization (e.g., Bandage) of the GFA graph files that were assembled from SPAdes 3.11.0 software (http://cab.spbu.ru/software/spades/, accessed on 25 December 2020) [24]. 2.3. Annotation and Analysis of C. esculentus Chloroplast DNA Sequence Annotation of C. esculentus chloroplast sequence was performed using PGA [25], and BLAST was used to evaluate the results. A circular gene map of the chloroplast genome was drawn using Organellar Genome DRAW v1.3.1 (https://chlorobox.mpimp-golm.mpg. de/OGDraw.html, accessed on 25 December 2020) [26]. The codon usage frequency and Diversity 2021, 13, 405 3 of 14 relative synonymous codon usage (RSCU) in C. esculentus were analyzed for all protein- coding genes (PCGs), using MEGAX to determine whether the chloroplast genes were under selection [27]. 2.4. Genome Comparison with Other Cyperus Species For comparative analysis, the chloroplast genome sequences of four Cyperus species, including C. fuscus Linn. (MK431855), C. glomeratus Linn. (MK423990), C. difformis Linn. (MK423991), and C. rotundus Linn. (MT473237), were retrieved from the Gen- Bank. The mVISTA program (http://genome.lbl.gov/vista/mvista/about.shtml, accessed on 12 November 2021) in Shuffle-LAGAN mode was used to compare the complete