Congrui Sun?Jie Li?Xiaogang Dai?Yingnan Chen

Introduction

Chloroplast(cp)genomes provide essential information for the study of biological processes in plant cells(Raubeson and Jansen 2005),such as biosynthesis of starch,fatty acids,pigments,and amino acids(Neuhaus and Emes 2000).It is generally accepted that cps originated from endosymbiosis of cyanobacteria(Timmis et al.2004).Cp genomes are typically paternally or biparentally inherited in gymnosperms(Reboud and Zeyl 1994).In contrast,cp genomes are maternally inherited in most angiosperms(Palmer et al.1988).

The chloroplast genomes of angiosperms have a typical quadripartite structure that contains a large single copy region(LSC)and a small single copy region(SSC)separated by two inverted repeat regions(IRs),and range from 120 to 160 kb in length with closed circular DNA(Sugiura 1995).Moreover,cp genomes are more conserved in genome structure and organization than nuclear and mitochondrial genomes(Raubeson and Jansen 2005).A study by Pyke(1999)revealed that approximately 400–1600 copies of cp genomes in each cell,which leads to high expression of cp genes.

In recent years,cp transformation has emerged as an environmentally friendly approach for plant genetic engineering(Daniell et al.2002).Foreign genes in transformed cps cannot be disseminated by pollen,because this plastid is maternally inherited in most flowering plants,thus posing significantly lower environmental risks.Cp transformation also has many other unique advantages over nuclear transformation,such as permitting the introduction of thousands of copies of foreign genes per plant cell,which allows uniformly and extraordinarily high expression levels of foreign genes,and eliminates gene silencing and the‘position effect’(Qian et al.2013;Daniell 2007;Verma and Daniell 2007).With the development of next-generation sequencing technologies,almost 1174 cp genomes in Viridiplantae have been completely sequenced and deposited in the NCBI Organelle Genome Resources(http://www.ncbi.nlm.nih.gov/genome/organelle/)to date.

Salix suchowensis(subgenus Vetrix)is a small,earlyflowering shrub willow endemic to China(Wang et al.1984)and mainly distributed in Jiangsu,Shandong,Zhejiang,and Henan Provinces in China(Fang et al.1999).For thousands of years,it has been used as basket-weaving material,but currently,it is being considered as a promising crop for bioenergy because of its high biomass yield(Smart and Cameron 2008).Because biomass yield is highly correlated with plant photosynthetic efficiency,analyzing and characterizing the cp genome of this shrub willow will provide essential information to improve productivity and facilitate the development of a plastid transformation system in this woody crop.

In 2014,the whole genome of S.suchowensis was sequenced by using a whole-genome shotgun strategy that incorporated Roche/454 and Illumina/HiSeq-2000 sequencing technologies,which produced 10.1 Gb 454 GS FLX reads and 230.2 Gb Illumina reads(Dai et al.2014).Because the sequencing libraries were constructed with leaf tissue,the generated reads included numerous sequence reads from the willow cp genome and provided sufficient sequence information for assembling the cp genome.In this study,our goals were to assemble and characterize the S.suchowensis cp genome by screening the organelle reads from the willow genome sequencing project and experimentally assessing the assembly quality of the cp genome.

Materials and methods

Sequence reads and cp genome assembly

Sequence reads were selected from the sequence database from the S.suchowensis genome sequencing generated by Dai et al.(2014).By mapping the raw reads to 660 cp genomes of terrestrial plants in the NCBI Organelle Genome Resources database(http://www.ncbi.nlm.nih.gov/genome/organelle/),we screened the willow cp sequence reads using BLASTN with an E value of 1e-50following the protocol described by Ma et al.(2016).The obtained reads were further assembled using Amos(Treangen et al.2011).Finally,a complete circular cp genome was established using Phrap(Ewing et al.1998)according to the reference cp genomes of S.purpurea(Wu 2015),Populus trichocarpa(Tuskan et al.2006),and Arabidopsis thaliana(Sato et al.1999).The complete circular cp genome of S.suchowensis was deposited in GenBank under accession no.KU341117.The sequencing depth was estimated using the modified formula of Zhang et al.(2010):the total size of the sampling reads divided by the size of the assembled cp genome.

Gene annotation and comparative analysis

First,the S.suchowensis cp genome was annotated using the online program Dual Organellar GenoMe Annotator(DOGMA,Wyman et al.2004).Genes that could not been annotated by DOGMA were manually identified by referring to the annotation of P.trichocarpa(Tuskan et al.2006).In addition,all tRNA genes were predicted with the online program tRNAscan-SE 1.21(Schattner et al.2005).Then,a circular cp genome map was generated using the OrganellarGenomeDRAW tool (OGDRAW) (http://ogdraw.mpimp-golm.mpg.de/).

Besides the sequenced S.suchowensis cp genome,eight complete cp genome sequences of Salicaceae species,including five poplars(Populus alba,AP008956;P.balsamifera,KJ664927;P.fremontii,KJ664926;P.tremula,KP861984;P.trichocarpa,EF489041;)and three willows(Salix babylonica,KT449800;S.interior,KJ742926;S.purpurea,KP019639),were obtained from NCBI Organelle Genome Resources database(http://www.ncbi.nlm.nih.gov/genome/organelle/). Comparative cp genome analysis among the nine Salicaceae species was performed by using blast 2.3.0(ftp://ncbi.nlm.nih.gov/blast/execu tables/blast+/2.3.0/).

cp genome structure and sequence analysis

Tandem repeats were evaluated using Tandem Repeat Finder 4.09(Benson 1999)with default settings.Forward repeats and palindromic repeats were identified using REPuter(http://bibiserv.techfak.uni-bielefeld.de/reputer/),and the minimal repeat size setting was greater than 30 bp with a Hamming distance of 3.Microsatellite or simple sequence repeats(SSRs)of one to six nucleotides were detected using the Perl script MISA(http://pgrc.ipk-gate rsleben.de/misa/),and thresholds of nine,five,five,three,three,and three repeat units were set for mono-,di-,tri-,tetra-,penta-and hexanucleotide SSRs,respectively.

Phylogenetic analysis

For phylogenetic analysis,besides S.suchowensis,we selected 31 rosid lineages that have complete cp genomes available.These lineages were from six rosid families(Salicaceae,Rosaceae,Moraceae,Fagaceae,Chrysobalanaceae,and Fabaceae),and Ginkgo biloba was used as the outgroup species.Based on the functional annotation from NCBI Organelle Genome Resources database,we identified 66 protein-coding genes that were commonly present in the analyzed cp genomes.These 66 orthologous genes were selected to construct the phylogenetic tree.The protein sequences were aligned with ClustalW 2.0(Larkin et al.2007),and a matrix consisting of 83,072 amino acids(aa)in default length was obtained.Optimal maximum likelihood(ML)and neighbor-joining(NJ)trees were constructed using MEGA 6.0(Tamura et al.2013).For the ML analyses,the Nearest-Neighbor-Interchange model was used with 1000 bootstrap replicates.The NJ tree was constructed using the Poisson model with 1000 bootstrap replicates.

Experimental verification of the cp genome assembly

To verify the assembly,we randomly designed 30 primer pairs(Table S1)based on the derived S.suchowensis cp genome using Primer Premier 5.0(Lalitha 2000).The cpDNA was extracted according to the method described by Mcpherson et al.(2013).Following amplification with these primers against the extracted DNA templates,the generated amplicons were sequenced on an ABI 3730 sequencer by Genscript Biology Company(Nanjing,Jiangsu,China).PCRs were performed as follows:each 20-μL PCR mixture consisted of 2.0 μL genomic DNA(100 ng),2.0 μL 10 × PCR buffer,0.2 μL Taq DNA polymerase(TaKaRa,Japan),1.6 μL MgCl2(25 mM),4.0 μL dNTP(2.5 mM each),1.0 μL of each primer(10 mmol/L),and 8.2 μL ddH2O.PCR amplification conditions included initial denaturation at 94°C for 4 min,followed by 30 cycles of 94 °C for 1 min,58 °C for 30 s,and 72 °C for 1 min,followed by a final extension at 72 °C for 5 min and storage at 4°C.

Results and discussion

cp genome assembly and structure analysis

By mapping the raw reads of the S.suchowensis genome sequencing project to the NCBI Organelle Genome Resources database(http://www.ncbi.nlm.nih.gov/genome/organelle/),a total of 1,171,821 reads(approximately 533 Mb)from the willow cp genome were obtained.De novo assembly by Amos(Treangen et al.2011)yielded 3773 contigs.Referring to the cp genomes of S.purpurea(Wu 2015),P.trichocarpa(Tuskan et al.2006),and A.thaliana(Sato et al.1999),these contigs were integrated into a complete circular pseudomolecule that was 155,508 bp long(GenBank accession no.KU341117).Thus,the sequencing depth of the cp genome was expected to be more than 3000×.The physical map of the derived cp genome(Fig.1)showed that it possessed a typical quadripartite structure that contained a pair of IRs(27,457 bp)separated by LSCs(84,385 bp)and SSCs(16,209 bp).

When we compared the cp genomes across nine Salicaceae species,we found that,although the cp genomes were more conserved in structure and organization than the nuclear and mitochondrial genomes are(Raubeson and Jansen 2005),the length of certain regions of the cp genomes varied among these closely related species.In these species,the length of IRs,LSCs,and SSCs ranged from 27,167 to 27,838 bp,84,377 to 85,979 bp,and 16,209 to 16,600 bp,respectively(Table 1),with very high sequence similarities.

The GC content,an important characteristic of the cp genome that affects genome stability(Yap et al.2015),in the cp genomes of Salicaceae species ranged from 36.65%to 37.00%,with an average of 36.73%(Table 1).The global GC content in the S.suchowensis cp genome was 36.73%,which was the same as the average of the closely related Salicaceae species,but higher than those of Wollemia nobilis(36.5%)(Yap et al.2015)and Metasequoia glyptostroboides(35.3%)(Chen et al.2015),and lower than those of Actinidia chinensis(37.2%)(Yao et al.2015),Macadamia integrifolia(38.1%)(Nock et al.2014),and Hyoscyamus niger(37.6%)(Sanchezpuerta and Abbona 2014).These species were more diverged from S.suchowensis than those listed in Table 1.

Fig.1 Physical map of Salix suchowensis complete chloroplast genome.Genes outside the circle are transcribed counterclockwise,and genes inside the circle are transcribed clockwise.Genes in the same color are in the same functional group.Internal circle of darker gray and lighter gray indicate GC content and AT content,respectively

Gene annotation

Annotation of the S.suchowensis cp genome revealed 143 genes.In the gene function analysis,the 143 genes were classified into four categories,including genes associated with self-replication,photosynthesis,and other functions,and genes of unknown function(Table 2).Among these genes,119 were unique,including four rRNA genes,30 tRNA genes,82 protein-coding genes,and three pseudogenes.Moreover,four rRNA,seven tRNA,and 13 proteincoding genes were duplicated in the IRs.Most of the unique genes contained no introns,but one intron wasfound in each of six tRNA genes and eight protein-coding genes,and two introns were found in each of three proteincoding genes(Table 2).

Table 1 Comparative analysis of cp genomes across nine Salicaceae species

Table 2 Summary of gene annotation for the cp genome of Salix suchowensis

ycf1 is one of the longest open reading frames in cp genomes and has been found in nearly all plastid genomes sequenced to date(Raubeson and Jansen 2005).Vries et al.(2015)assumed that ycf1 encodes the translocon on the inner envelope of chloroplasts(TIC).Drescher et al.(2000)predicted that ycf1 is involved in essential pathways in cellular metabolism or serves a structural function for the plastid compartment.The function of ycf1 has not been clearly resolved;nevertheless,it is considered essential to plant survival(Drescher et al.2000).

In the sequenced cp genome,ycf1 usually spans the boundary of the IR and SSC regions(Raubeson and Jansen 2005).Based on the common location of ycf1 in the plastid genome,a copy of the ycf1 gene(5424 bp)was found at the IRa/SSC border(Fig.1),and a truncate copy of a ycf1 pseudogene(1878 bp)was present at the IRb/SSC border(Fig.1)in the S.suchowensis cp genome.ycf1 is highly variable and approximately 5500 bp in plant plastid genomes.Compared with Chlorophyta species,the length of the S.suchowensis YCF1 protein(1807 aa)is much longer than that of Nephroselmis olivacea(956 aa;NC_0000927),and much shorter than that of Schizomeris leibleinii(3212 aa;NC_015645).

Recent studies demonstrated that genes can transfer from the cp genome to the nuclear genome at a relatively high frequency(Huang et al.2003;Stegemann and Bock 2006).infA,which encodes the plastid translation initiation factor 1,provides a striking example of gene transfer events(Millen et al.2001).We found a parallel of infA gene with an uncommon initiation codon of AGA in the S.suchowensis cp genome that was located in the LSC,and the length of this gene was 165 bp.Sequence alignment detected a fragment with high similarity(92.73%)on chromosome II of the S.suchowensis nuclear genome(Fig.2).This infA-like fragment might be transferred from the cp genome to the nuclear genome.

Repeat sequence analysis

Previous studies revealed that gene duplication,gene expansion,and cpDNA rearrangement seemed to be associated with repetitive sequences(Cavalier-Smith 2002).We identified 31 tandem repeats,16 forward repeats,and five palindromic repeats in the(Table S2).The tandem repeat units were 7–26 bp long,and almost all the tandem repeats were located at intergenic spacer(IGS)regions except for one located in an intron region.Additionally,the forward repeat units were 30–76 bp long.The majority of these repeats were distributed in IGS regions,with some of them detected in protein-coding and tRNA gene regions.Alternatively,for the palindromic repeats,the repeat units were 30–42 bp long.Four repeats were detected in IGS regions,and one was located in a tRNA gene region.Overall,21 repeats≥30 bp were detected in the S.suchowensis cp genome,and most(84.6%)were distributed in the intergenic spacer region.These repeat motifs can be selected for population studies since they are an informative source for developing markers.

Microsatellites or SSRs are common in the plant cp genome.The MISA output revealed 148 perfect SSRs in S.suchowensis cp genome.Among these,126 SSRs were mononucleotide repeats,10 were dinucleotide repeats,11 were tetranucleotide repeats,and one was a pentanucleotide repeat(Table S3).Among the monomers,121 consisted of A/T repeats,and only five consisted of G/C repeats.The A/T content of monomers was similar to that in the M.glyptostroboides cp genome(96.03%)(Chen et al.2015).All dinucleotides in the S.suchowensis cp genome were AT/TA repeats,and A/T contents in tetramers and pentamers were 86.36%and 80%,respectively.When analyzed with the same parameters in MISA,the average SSR length and SSR density in S.suchowensis cp genome(10.23 bp,9.74/1000 bp)were found to be lower than those of the W.nobilis(16.97 bp,14.65/1000 bp)(Yap et al.2015)and M.glyptostroboides cp genomes(11.01 bp,9.85/1000 bp)(Chen et al.2015).

Chloroplast SSRs(cpSSR)represent ideal complementary molecular tools for nuclear genetic markers.In combination with nuclear SSR markers,cpSSR markers have a high capability in differentiating among closely related taxa,e.g.,grapes(Arroyo-Garcia et al.2006).In the S.suchowensis cp genome,most SSRs were AT-rich,and the mononucleotides were found to be the dominant repeats.These results are consistent with the previous contention that cpSSRs are generally composed of short polyA or polyT repeats(Kuang et al.2011).

IR contraction and expansion

Fig.2 Sequence alignment of infA from cp genome and that from nuclear genome.a cp genome of S.suchowensis;b nuclear genome of S.suchowensis

IRs are prominent features of most angiosperm cp genomes.During the evolutionary process of angiosperms,IR contraction and expansion might influence cp genome size(Goulding et al.1996;Wang et al.2008)and could create pseudogenes that cannot be transcribed(Wang et al.2008).Here,we compared in detail the IR/single copy(SC)boundaries of four rosid plants:S.suchowensis,S.integra,Prunus padus and Morus notabilis(Fig.3).

In the cp genome of S.suchowensis and S.integra,the IRb/LSC junction was found within the rpl22 gene,and the rpl22 pseudogene(52 bp)was detected at the IRa/LSC boundary,whereas the rps19 pseudogene(39 bp)was found at the IRa/LSC border in P.padus.As for M.notabilis,IRb was found to be immediately adjacent to the rps19 gene,and no pseudogene was observed at the IRa/LSC boundary.The trnH genes were all located within the LSC region in these four species,but varied in being between 16 and 36 bp from the IRa/LSC junctions.

In most land plant cp genomes,the ycf1 pseudogene and ndhF are located at the LSC/IR border,such as in P.trichocarpa(Tuskan et al.2006),Glycine stenophita(Sherman-Broyles et al.2014),and S.miltiorrhiza(Qian et al.2013).At the IRa/SSC border of the four cp genomes,the IR expanded into the ycf1 gene,creating the ycf1 pseudogene at the IRb/SSC border.The length of the ycf1 pseudogene was 1878 bp in S.suchowensis,1713 bp in S.integra,1036 bp in P.padus,and 1002 bp in M.notabilis.In addition,the ycf1 pseudogene and ndhF gene overlapped in S.suchowensis,P.padus and M.notabilis by 140 bp,19 bp and 26 bp,respectively.

Phylogenetic trees

To elucidate the phylogenetic position of S.suchowensis among rosids,we analyzed 66 orthologous protein-coding genes present in the cp genomes of 33 species(Table S4).The ML bootstrap analysis resolved 29 nodes,of which 25 had bootstrap values≥90%,and 18 of these had bootstrap support of 100%(Fig.4).With the NJ tree,we obtained a sum of branch lengths of 0.61439509.The NJ bootstrap analysis was similar to that of the ML tree that resolved into 29 nodes,because 24 nodes had bootstrap values≥90%,and 20 of these had 100%bootstrap support(Fig.S1).Both the ML and NJ trees showed that these species were evident in three rosid categories:Rosids I(Salicaceae and Chrysobalanaceae),Rosids II(Fagaceae,Moraceae,and Rosaceae),and Rosids III(Fabaceae).In Rosids I,S.suchowensis and S.babylonica were the closest relatives.The topology of the derived ML tree was very similar to that of the established NJ tree;the only incongruence was the position of P.fremontii and of P.balsamifera relative to P.trichocarpa.It is noteworthy that the bootstrap supports for grouping P.fremontii or P.balsamifera with P.trichocarpa are relatively low in both the ML and NJ trees.The relationships of these three species might not be able to be properly resolved merely based on plastid-level information.

Verification of the S.suchowensis cp genome assembly

In this study,the raw reads were generated by next-generation sequencing platforms,and the screening of reads and cp genome assembly were conducted merely by using bioinformatics tools.To verify the quality of the assembly,we sequentially selected 30 sites from the cp genome.

Fig.3 Comparison of the borders of IR regions among the cp genomes of four rosid plants.Three colors were used to indicate the LSC,IR and SSC regions,respectively.The figure mainly indicates the shift of the genes located in the IR border. ‘ψ’means pseudogene,‘overlap’means overlap of ψycf1 and ndhF

Fig.4 Maximum likelihood(ML)phylogenetic tree

All synthesized primers succeeded in PCR amplification.Sequenced using a Sanger sequencer,the 30 amplicons covered a total physical length of 18,639 bp.Alignment of the amplicon sequences to the genome assembly produced sequence errors in seven of the tested sites,whereas a 100%match was revealed with amplicons at the other 23 sites in the cp genome assembly.The overall accuracy of the derived assembly was estimated to be 99.88%.Therefore,the cp genome obtained in this study was high quality.Moreover,as mentioned above,we detected raw reads that covered over 3000×sequencing depth of the S.suchowensis cp genome.The high sequencing depth ensures accuracy and integrity of the obtained pseudomolecule of the cp plastid.

Conclusion

We revealed a pseudomolecule of the S.suchowensis cp genome with high reliability based on raw reads generated by next-generation sequencing platforms.The genome structure and organization of the S.suchowensis cp genome were similar to those in other Salicaceae species.IR expansion was observed in S.suchowensis in comparison with those of other rosid plants.The repeats and SSRs identified here will be informative sources for developing markers for evolutionary and population-genetic studies.The S.suchowensis cp genome obtained in this study is highly desirable for facilitating the biological study of this promising biofuel plant and will facilitate more extensive studies such as cpSSR development,endosymbiotic gene transfer and plastid genetic engineering in willows.

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