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Research Note *Corresponding author Wentao Sheng, Department of Biological Technology, Nanchang Normal University, Nanchang 330032, The Complete Chloroplast Jiangxi, , Tel: 86-0791-87619332; Fax: 86-0791- 87619332; Email: Submitted: 14 September 2017 Genome Sequence of Accepted: 09 October 2017 Published: 10 October 2017 (Asparagus ISSN: 2333-6668 Copyright © 2017 Sheng et al. officinalis L.) and its OPEN ACCESS

Keywords Phylogenetic Positon within • Asparagus officinalis L • Chloroplast genome • Phylogenomic • Asparagales Wentao Sheng*, Xuewen Chai, Yousheng Rao, Xutang, Tu, and Shangguang Du Department of Biological Technology, Nanchang Normal University, China

Abstract Asparagus (Asparagus officinalis L.) is a horticultural homology of medicine and food with health care. The entire chloroplast (cp) genome of asparagus was sequenced with Hiseq4000 platform. The complete cp genome maps a circular molecule of 156,699bp built with a quadripartite organization: two inverted repeats (IRs) of 26,531bp, separated by a large single copy (LSC) sequence of 84,999bp and a small single copy (SSC) sequence of 18,638bp. A total of 112 genes comprising of 78 protein-coding genes, 30 tRNAs and 4 rRNAs were successfully annotated, 17 of which included introns. The identity, number and GC content of asparagus cp genes were similar to those of other asparagus genomes. Analysis revealed 81 simple sequence repeat (SSR) loci, most composed of A or T, contributing to a bias in base composition. A maximum likelihood phylogenomic evolution analysis showed that asparagus was closely related to cyrtonema that belonged to the Asparagales. The availability of the complete cp genome sequence of asparagus provides valuable information for chloroplast genetic engineering and phylogenetic analyses in Asparagales.

ABBREVIATIONS And Asparagus officinalis L. has been used as model for CP: Chloroplast; IRs: Inverted Repeats; LSC: Large Single sexcompound, determination which is and wide differentiation spreadly cultivated due to the in presenceChina [3,4]. of Copy; SSC: Small Single Copy; SSR: Simple Sequence Repeat; APG: both dioecious and and rodioecious plants within population Angiosperm Phylogeny Group; DOGMA: Dual Organellar Genome Annotator; ML: Maximum Likelihood Asparagus officinalis L. The asparagus plants are divided into the [5]. However, the taxonomic boundaries are not well defined for INTRODUCTION method [6] but in the Angiosperm Phylogeny Group (APG Compared with the nuclear genome, chloroplast (cp) is a ma- , in the traditionalAsparagus morphological classification jor semiautonomous organelle, with obvious characteristics of relatively conserved size, multicopies, stable gene content and I) classification system, the is classified into the structure, a maternal inheritance manner, a moderate nucleotide Asparagaceae ,only which includes was divided asparagus into one plants, individual and it was family included [7] in evolution rate with protein-coding genes, and so on [1]. The com- the2003 followingrevision version branches of APG of IIAsparagaceae classification,, strictAphyllanthaceae definition of, parison of complete cp genome sequences is becoming increas- Laxmanniaceae, Hyacinthaceae, Hesperocallidaceae, Ruscaceae ingly important for the study of reconstructing the evolutionary the genus and its subdivisions are still unresolved questions inplants the in the wide of sense Asparagaceae [8]. Furthermore,. Therefore, the classification availability of relationships, classification and genetic diversity of plants [2]. Up to NCBI (https://www.ncbi.nlm.nih.gov/genomes/Genomes- a complete A. officinalis L. cp genome sequence is crucial to to date, the number of 2113 cp genomes of green plants uploaded ) has been available until Aug understand phylogenetic relationships among major lineages of Asparagaceae and facilitates asparagus genetic improvement. Group.cgi?taxid=2759&opt= 16th,Asparagus 2017. officinalis L. is a perennial herb with functional These problems would be expected to be solved by

Cite this article: Sheng W, Chai X, Rao Y, Tu X, Du S (2017) The Complete Chloroplast Genome Sequence of Asparagus (Asparagus officinalis L.) and its Phy- logenetic Positon within Asparagales. Int J Plant Biol Res 5(4): 1075. Sheng et al. (2017) Email:

Central Bringing Excellence in Open Access Phylogenetic analysis sequence and cp genomic information available for asparagus, phylogenetic analysis of cp genome. However, there is little Forty-two species representing the Asparagales of angiosperm were sampled to reconstruct a phylogeny of study, we reported the complete chloroplast genome sequence Asparagales with cp coding-protein genes. The cp genome of ofwhich posed a barrier to L.current (deposited studies into in these Gene areas bank [9]. accession In this Asparagus officinalis brownii var. viridulum and sativa L. were used as the out group. There were altogether seventy-seven genes shared sequencing data. by all forty-four cp genomes. Each gene from each genomic number: NC_034777.1) from the whole-genome high-throughput MATERIALS AND METHODS W. The resulting gene alignments were assembled into a data DNA extraction and sequencing matrixsequence for was each modified genome. manually Optimal and was were aligned conducted using Clustal with Total genomic DNA was extracted from 1 g of fresh Maximum likelihood (ML) analyses using in Phylogenetic Analyis from Asparagus officinalis L. cv. Atlas plant (male) cultivated in appropriate evolution model was determined implemented in the greenhouse of Jiangxi Agricultural Science Academy from Using Parsimony (PAUP) version 4.0b10 (Swofford, USA). The ° °

The genome libraries was constructed and sequenced on the theRESULTS Model 3.7 AND software. DISCUSSION Nanchang (E115 27′,N28 09′), using mCTAB method [10]. Genome content and organization in Asparagus manufacturer’s instructions with multiplexing on HiSeq4000 officinalis L. Chloroplastflow cell lanes (Illuminagenome Inc.).assembly and annotation The complete cp genome of Asparagus officinalis For the genome, the raw reads were assembled into non- bp in size and exhibits a typical circular structure including a pair is 156,699 redundant contigs with SPAdes 3.6.1 and SOAPdenovo2, a de novo of inverted repeats (IRs) of 25,783 bp separated the genome into ofsequence the cp genome assembly was software screened package, by Blast with program k=30 and for eachscaffolding of the two single-copy regions (large single copy, LSC 82,212 bp; small splicedcontigs contig,having anda minimum the cp genome length contig of 100 was bp assembled[11,12]. The by contig using single copy, SSC 16,670 bp; (Figure1). Coding regions (91,260 http://www.genecodes.com bp), including protein-coding genes (79,413 bp), tRNA genes reads were mapped to the spliced cp genome sequence to verify (2,801 bp), and rRNA genes (9,046 bp), account for 60.66% of the Sequencer 5.4.6 ( ) [13]. Then all the genome, while noncoding regions (59,188 bp), including introns (17,750 bp) and intergenic spacers (41,438 bp), account for the remaining 39.34% of the genome. The overall A+T content of the tothe twelve correctness large contigs of the splicedwith additional contig used Sanger by sequencing,Geneious 8.1 and [14]. to whole genome is 62.24%. verifyPrimer the walking junctions were between then used the to single-copy fill the gaps and between the IRs the regions. seven There are a total of 136 genes predicted in the genome, of Chloroplast genome annotation which 112 are unique, including 78 protein-coding genes, 30 Genome annotation was accomplished using the Dual intRNA the genes,IR regions, and 4however, ribosomal only RNA a part genes of ycf1(Figure gene 1) is andduplicated (Table Organellar Genome Annotator (DOGMA) software to annotate the in1). the Ten junction protein ofcoding, SSC and 8 tRNA IRA regions. and 4 rRNA According genes toare its duplicated function, genes encoding proteins, transfer RNAs (tRNAs), and ribosomal

subunits,the predicated rRNA, genes the vast are majoritydivided intoof tRNA three gene, categories. associated The withfirst RNAs (rRNAs) [15].http://lowelab.ucsc.edu/tRNAscan-SE All of the identified tRNA genes). were The category owns a total of 60 genes including RNA polymerase genomefurther verified map of Asparagususing the corresponding officinalis L. was structures drawn by predicted Organellar by related with photosythesis, including large subunit gene, GenometRNAscan-SE DRAW 1.21( http://ogdraw.mpimp-golm.mpg.de/index. thetranscription photosynthetic and translation; electron transport the second chain category components has 47 genes Shtml [16]. third kinds are associcted with amino acids, fatty acids and other Repeat structure finding substancesand the presumed biosynthesis NAD(P)H related dehydrogenase genes, as well subunitas some gene;unknown the Microsatellite (mono-, di-, tri-, tetra-, penta-, and function genes (Table 1). The infA, ycf1, ycf68 are pseudogenes hexanucleotide repeats) detection was performed by using judging by the presence of several terminal codons in the coding Website (http://wsmartins.net/websat/) and MISA(http://pgrc. regions. ipk-gatersleben.de/misa/) with thresholds of ten repeat units for three repeat units for tetra-, penta- and hexanucleotide. There are 17 introns containing genes (12clpP protein-coding, ycf3, rps12) mononucleotide, five repeat units for di- and tri-nucleotide, and and 5 tRNAs), among which 9 protein-codingrps12 is a special genes trans-splicing and 5 tRNAs Comparative chloroplast genomic analysis contain a intron, and 3 protein-coding genes ( With the cp genome of Asparagus officinalis L. as a reference, exonhave situatedtwo introns in the (Table IR region. 2). And four species (Asparagus officinalis L., Polygonatum sibiricum, gene, with its 5’-end exon located in the LSC region, and 3’-end Distribution characters of SSRs in chloroplast genome americana, asphodeloides) were compared to assess the variability of the cp genome. There were three microsatellites including full

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Figure 1 Chloroplast genome map of Asparagus officinalis L.. The genes inside and outside of the circle are transcribed in the clockwise and counter clockwise directions, respectively. Genes belonging to different functional groups are shown in different colors. The thick lines indicate the extent of the inverted repeats (IRa and IRb) that separate the genomes into small single-copy (SSC) and large single-copy (LSC) regions. microsatellites, incomplete microsatellites and composite Phylogenetic analysis of Asparagales species using cp microsatellites types in the genome, of which the number was genome sequences was mainly composed of forty mono-base and eleven bi-base, The phylogenetic relationships of Asparagus officinalis in 69, 1 and 11, respectively. In the full type microsatellites, it Asparagales. accounting for 49.38% and 13.38% of the total number. In the plants were reconstructed, based on 77 protein- mono-base repeats, the number of A and T were 13 and 25, coding genes sequences obtained from 42 taxa of The total aligned length of these genes was 754,72 characters. typesaccounting in cpSSR for of 18.8% Asparagus and 36.2%,cp genome. while In Cthe and bi-base G accounted repeats, forAT Asparagus only 2.8% (Table 3), which showed that A and T repeat were main officinalisMaximum and likelihood Polygonatum (ML) analysis cyrtonema was formed performed as withsister PAUP taxa version 4.0b10.The ML strongly supported (6) and TA type (2) account for 8.7% and 2.8%, respectively. And Asparagus officinalis and evolved from separate with a pp value of 100 % (Figure 2). But the relationship of mostlythe number composed of tri-base, of low fourrepeat bases, number, and contributingfive bases types to a biaswas of5, monophyletic , repestively. Therefore, the phylogenetic Asparagus12 and 1, respectively. officinalis cp Therefore,genome. it was concluded that SSR loci position within Asparagales from the cp genome data didn’t Comparison with other cp genomes in genome content and organization in Asparagales support the traditonal classification system of Liliaceae [17], but it was proved to accord with the new classification APG system The genomic features of Asparagus officinalis and [8,18,19].CONCLUSION those of three species from other families were compared In summary, this study reported the whole cp genome with Asparagales , including Polygonatum sibiricum sequence of Asparagus officinalis. And the analysis of genome , , Anemarrhena structure and sequence showed that it was nearly identical to asphodeloides (NC_029485) (KX519714) those species in Asparagales. coding genes in cp genome indicated that Asparagus officinalis of Asparagus officinalis(KX931449). were The similar sequence to those length of other(156699 species bp), was much more closely relatedThe to phylogeneticPolygonatum evolutioncyrtonema of, but 77 thewithin GC thecontent Asparagales (37.76%) and the gene content and arrangement not to Lilium distichum in Liliaceae. (Table 4). Int J Plant Biol Res 5(4): 1075 (2017) 3/6 Sheng et al. (2017) Email:

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Figure 2

Maximum likelihood (ML) analysis inferred from 77 genes protein coding genes from 42 taxa. Table 1: Gene contents of complete chloroplast genome of Asparagus officinalis L. Category for genes Group of gene Name of gene Photosynthesis related genes large subunit of Rubisco rbcL subunits of photosystem Ⅰ psaA,psaB,psaC,psaI,psaJ

assembly/stability of photosystem a Ⅰ ycf3 ,ycf4 psbA,psbB,psbC,psbD,psbE,psbF,psbH,psbI,psbJ,psbK,psbL,psbM,psbN,psb subunits of photosystem Ⅱ T,psbZ subunits of ATP synthase atpA, atpB, atpE, atpFa, atpH, atpI cytochrome b/f compelx petA, petBa, petDa, petG, petL, petN c-type cytochrome synthesis ccsA

ndhAa, ndhBa,b, ndhC, ndhD, ndhE, ndhF ,ndhG, ndhH, ndhI, ndhJ, ndhK dehydrogenase Transcription and translation subunits of NADPH DNA dependent RNA polymerase rpoA, rpoB, rpoC1a, rpoC2 related genes rps2, rps3, rps4, rps7b, rps8, rps11, rps12a,b, rps14,rps15, rps16a, rps18, ribosomal proteins rps19,rpl2a,b, rpl14, rpl16a, rpl20, rpl22, rpl23b, rpl32, rpl33,rpl36 translation initiation factor infA RNA genes ribosomal RNA rrn4.5b, rrn5b, rrn16b, rrn23b trnA-UGCa,b, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-UCC, trnG- GCCa, trnH-GUGb, trnI-CAUb, trnI-GAUa,b,trnK-UUUa, trnL-CAAb, trnL-UAAa, transfer RNA trnL-UAG, trnfM-CAU,trnM-CAU, trnN-GUUb, trnP-UGG, trnQ-UUG,trnR- ACGb, trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU,trnT-UGU, trnV- GACb, trnV-UACa, trnW-CCA, trnY-GUA Other genes muturase matK Envelop membrane protein cemA ATP-dependent protease subunit clpPa p gene Genes of unknown function conserved open reading frames ycf1, ycf2b aGenes containing introns; bduplicated gene (genes present in the IR regions).

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Table 2: Characteristics of genes including introns and exons in the cp genomes of Asparagus officinalis L. Gene Exon I Intron I Exon II Intron II Exon III trnA-UGCc trnG-UCC 38 817 35 trnI-GAUc 24 675 48 trnK-UUU 36 943 37 trnL-UAA 35 2586 37 trnV-UAC 567 529 50 rps16 861 35 598 39 rpl2c 666 209 40 rpl16 431 391 atpF 399 386 9 petB 6 411 840 144 petD 8 753 642 ndhA 736 481 ndhBc 540 1127 558 ycf3 755 701 777 clpP 152 743 229 721 126 rpoC1 252 667 292 805 71 cGenes located in the inverted repeats. 1625 751 436

Table 3: Types of complete repeat copy of cpSSR in asparagus cpDNA. Base repeat type cpSSR motif cpSSR number Ratio in total (%) Mono- A 18.8 T 13 C 25 36.2 Bi- AT 6 2 2.8 TA 8.7 TC 2 2.8 GA 1 2 2.8 Tri- ATA 1 1.4 ATT 1.4 CTC 1 2 2.9 TTC 1 1.4 1.4 Table 4: Comparison of general features of the cp genomes in Asparagales. Anemarrhena Genome features Asparagus officinalis L. Polygonatum sibiricum Agave americana asphodeloides Total length(bp) LSC length(bp) 156699 152960 157274 156867 SSC length(bp) 84999 81470 86060 85039 IRs(bp) 18638 18519 19500 19846 Total genes 53062 52971 51714 51982 Protein genes 81 112 115 108 112 rRNA genes 78 74 78 tRNA genes 4 4 4 4 30 30 30 30 GC content(%) 37.76 37.95 37.84 37.85 ACKNOWLEDGEMENTS We thank Dr. Guangyu Chen and the Innovative Team of (GJJ161243), Doctoral research project of Nanchang Normal Asparagus officinalis L, Jiangxi Academy of Agricultural Sciences, University (NSBSJJ2015036), and the program of the molecular breeding innovation team of Nanchang Normal University (090150000114).REFERENCES Nanchang 330000, China for providing of asparagus. We 1. Gao L, Su YJ, Wang T. Plastid genome sequencing, comparative technologyalso acknowledge project financialof Jiangxi supportsProvincial by Department Natural science of Education youth genomics, and phylogenomics: current status and prospects. J Syst foundation project of Jiangxi (20171BAB214024), Science and

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Cite this article Sheng W, Chai X, Rao Y, Tu X, Du S (2017) The Complete Chloroplast Genome Sequence of Asparagus (Asparagus officinalis L.) and its Phylogenetic Positon within Asparagales. Int J Plant Biol Res 5(4): 1075.

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