Conservation Genet Resour (2017) 9:569–571 DOI 10.1007/s12686-017-0724-2

TECHNICAL NOTE

Characterization of the complete chloroplast genome of the wild Himalayan Pyrus pashia (: : Maloideae)

Lu Bao1 · Ke Li1 · Yuanwen Teng2 · Dong Zhang1

Received: 27 February 2017 / Accepted: 10 March 2017 / Published online: 25 March 2017 © Springer Science+Business Media Dordrecht 2017

Abstract Pyrus pashia is a deciduous fruiting tree spe- valleys with an elevation of 600–3000 m (Lu et al. 2003). cies of high commercial, scientific, nutritional and thera- This possesses high commercial, scientific, nutri- peutic values. Many of its wild populations are in danger, tional and therapeutic values. It has been widely used as and need urgent conservation. To contribute to such efforts, an important germplasm resource for genetic breeding of its complete chloroplast genome was assembled from as well as a pear rootstock across its geographic range high-throughput Illumina sequencing data. The circular because of its high adaptation to local environments (Liu genome is 160,153 bp long, and displays a typical quadri- et al. 2013; Lu et al. 2003). It is also well-known for its partite structure of the large (LSC, 88,129 bp) and small nutritional and therapeutic importance, and has long been (SSC, 19,252 bp) single-copy regions, separated by a pair used conventionally by many communities in the Himala- of inverted repeat regions (IRs, 26,386 bp each). It harbors yas for treating gastrointestinal, respiratory and cardiovas- 112 gene species, including 78 protein-coding, 30 transfer cular ailments (reviewed by Janbaz et al. 2015). In addition, RNA and 4 ribosomal RNA gene species. Being similar to it is regarded as an intermediate species between the occi- many other confamilial taxa, it has a biased base composi- dental and oriental pear groups, and thus may have played tion (31.35% A, 18.63% C, 17.93% G and 32.09% T) with an important role in the evolution of the genus Pyrus (Chal- an overall A+T content of 63.44%. Phylogenetic analysis lice and Westwood 1973; Rubtsov 1944). Despite these sig- suggested that P. pashia was more closely related to its con- nificant values, many of its wild populations are in danger geners than to the other confamilial taxa with sequenced due to the ecological damages caused by global warming chloroplast genomes. and urbanization, and need urgent conservation (Liu et al. 2013). To gain a better insight into its genetics and genom- Keywords Pyrus pashia · High-throughput sequencing · ics and thus contribute to its conservation, we assembled Chloroplast genome · MITObim its complete chloroplast genome by using high-throughput Illumina sequencing technology. The annotated genomic sequence has been submitted to GenBank with the acces- The wild Himalayan pear Pyrus pashia is a deciduous fruit- sion number KY626169. ing tree species within the family Rosaceae, and is native to Total genomic DNA was extracted from fresh leaves of a Southwest and the , naturally occurring in single individual with the DNeasy Plant Mini Kit (Qiagen, Valencia, CA), and was used for the shotgun library con- * Dong Zhang struction and the subsequent high-throughput sequencing [email protected] on the Illumina HiSeq 2500 Sequencing System (Illumina,

1 CA, USA). In total, 16.03 M of 125-bp raw paired reads College of Horticulture, Northwest A&F University, were yielded, quality-trimmed with CLC Genomics Work- 3 Taicheng Road, Yangling 712100, Shaanxi, People’s Republic of China bench v9.0 (CLC Bio, Aarhus, Denmark), and then used for the assembly of chloroplast genome with MITObim v1.8 2 College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang, (Hahn et al. 2013), with that of (GenBank: People’s Republic of China AP012207) (Terakami et al. 2012) as the initial reference.

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The genome annotation was done in Geneious R9 (http:// all the others completely within the IR regions. Sixteen www.geneious.com; Biomatters Ltd., Auckland, New Zea- gene species (atpF, ndhA, ndhB, petB, petD, rpl16, rpl2, land) by aligning with the chloroplast genomes of such rpoC1, rps12, rps16, trnA-UGC, trnG-UCC, trnI-GAU, confamilial taxa as Pyrus pyrifolia (AP012207) (Terakami trnK-UUU, trnL-UAA and trnV-UAC) contain a single et al. 2012), Pyrus spinosa (HG737342) (Korotkova et al. intron, while another two PCG species (clpP and ycf3) 2014) and Prunus yedoensis (KP732472) (Cho et al. 2016). harbor a couple of introns. The base composition is biased A physical map of the genome was generated by using the (31.35% A, 18.63% C, 17.93% G and 32.09% T) with an web server OGDRAW (http://ogdraw.mpimp-golm.mpg. overall A+T content of 63.44%, which is similar to many de/) (Lohse et al. 2013). other taxa within the family Rosaceae, e.g. Pyrus pyrifolia The chloroplast genome of P. pashia is 160,153 bp long, (AP012207) (Terakami et al. 2012) and Prunus yedoensis and displays a typical quadripartite structure of the large (KP732472) (Cho et al. 2016). The overall A+T content is (LSC, 88,129 bp) and small (SSC, 19,252 bp) single-copy higher than those of the IR regions (57.35%), but is lower regions, separated by a pair of inverted repeat regions (IRs, than those of the LSC (65.75%) and SSC (69.60%) regions. 26,386 bp each) (Fig. 1). In total, 112 gene species were To ascertain its phylogenetic status within the family annotated, including 78 protein-coding (PCG), 30 transfer Rosaceae, a neighbor-joining (NJ) tree was reconstructed RNA (tRNA) and four ribosomal RNA (rRNA) gene spe- using the coding sequences of 77 PCGs for a group of 20 cies. Most gene species occur as a single copy, except for species with MEGA6 (Tamura et al. 2013) (Fig. 2). All 19 of them with double copies, including eight PCG spe- 20 species were clustered into two monophyletic groups, cies (ndhB, rpl2, rpl23, rps7, rps12, rps19, ycf1 and ycf2), which corresponded to the two subfamilies Maloideae and seven tRNA species (trnA-UGC, trnI-CAU, trnI-GAU, Rosoideae within the family Rosaceae. As expected, P. trnL-CAA, trnN-GUU, trnR-ACG and trnV-GAC) and the pashia is more closely related to its congeners (P. pyrifolia four rRNA species (rrn4.5, rrn5, rrn16 and rrn23). Out and P. spinosa) than to the other confamilial taxa (Pentac- of these 19 gene species, three species (rps19, ycf1 and tina, Prinsepia and Prunus). rps12) are partially located within the IR regions, while

Fig. 1 Physical map of the Pyrus pashia chloroplast genome. Genes shown outside the outer circle are transcribed in the clockwise direction, whereas those inside are tran- scribed in the counterclockwise direction. Areas dashed light and darker gray in the inner circle indicates the A+T and G+C contents of the genome, respectively

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Fig. 2 Phylogeny of 20 species within the family Rosaceae based on the neighbor-joining (NJ) analysis of the concat- enated coding sequences of 77 chloroplast PCGs. The bootstrap values were based on 500 repli- cates, and are shown next to the branches

Acknowledgements This work was supported by the National Nat- genomic regions is lineage-specific: implications of pairwise ural Science Foundation of China (31501741), the Special Financial genome comparisons in Pyrus (Rosaceae) and other angiosperms Grant from the China Postdoctoral Science Foundation (2014T70939) for marker choice. PLoS ONE 9:e112998. doi:10.1371/journal. and the Shaanxi Postdoctoral Science Foundation (133782). pone.0112998 Liu J, Sun P, Zheng X, Potter D, Li K, Hu C, Teng Y (2013) Genetic structure and phylogeography of Pyrus pashia L. (Rosaceae) in Yunnan Province, China, revealed by chloroplast DNA analyses. References Tree Genet Genom 9:433–441. doi:10.1007/s11295-012-0564-x Lohse M, Drechsel O, Kahlau S, Bock R (2013) OrganellarGenome- Challice JS, Westwood MN (1973) Numerical taxonomic studies of DRAW—a suite of tools for generating physical maps of plastid the genus Pyrus using both chemical and botanical characters. and mitochondrial genomes and visualizing expression data sets. Bot J Linn Soc 67:121–148. doi:10.1111/j.1095-8339.1973. Nucl Acids Res 41:W575–W581. doi:10.1093/nar/gkt289 tb01734.x Lu L, Gu C, Li C, Jiang S, Alexander C, Bartholomew B, Brach AR, Cho M-S, Hyun Cho C, Yeon Kim S, Su Yoon H, Kim S-C (2016) Boufford DE, Ikeda H, Ohba H, Robertson KR, Spongberg SA Complete chloroplast genome of Prunus yedoensis Matsum. (2003) Rosaceae. In: Flora of China Editorial Committee (ed) (Rosaceae), wild and endemic flowering cherry on Jeju Island, Flora of China, vol 9. Science Press/Missouri Botanical Garden Korea. Mitochondr DNA A 27:3652–3654. doi:10.3109/194017 Press, Beijing/St. Louis, pp 46–434 36.2015.1079840 Rubtsov GA (1944) Geographical distribution of the genus Pyrus Hahn C, Bachmann L, Chevreux B (2013) Reconstructing mitochon- and trends and factors in its evolution. Am Nat 78:358–366. drial genomes directly from genomic next-generation sequencing doi:10.1086/281206 reads—a baiting and iterative mapping approach. Nucl Acids Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) Res 41:e129. doi:10.1093/nar/gkt371 MEGA6: molecular evolutionary genetics analysis version 6.0. Janbaz KH, Zaeem Ahsan M, Saqib F, Imran I, Zia-Ul-Haq M, Abid Mol Biol Evol 30:2725–2729. doi:10.1093/molbev/mst197 Rashid M, Jaafar HZE, Moga M (2015) Scientific basis for Terakami S, Matsumura Y, Kurita K, Kanamori H, Katayose Y, use of Pyrus pashia Buch.-Ham. ex D. Don. fruit in gastroin- Yamamoto T, Katayama H (2012) Complete sequence of the testinal, respiratory and cardiovascular ailments. PLoS ONE chloroplast genome from pear (Pyrus pyrifolia): genome struc- 10:e0118605. doi:10.1371/journal.pone.0118605 ture and comparative analysis. Tree Genet Genom 8:841–854. Korotkova N, Nauheimer L, Ter-Voskanyan H, Allgaier M, Borsch doi:10.1007/s11295-012-0469-8 T (2014) Variability among the most rapidly evolving plastid

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