{'fraction_non_alphanumeric': 0.03593266918584679, 'fraction_numerical': 0.030436276193747853, 'mean_word_length': 4.9218877135882835, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 2, 'lorem ipsum': 0, 'www.': 2, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Development of PCR-based markers for discriminating Solanum berthaultii using its complete chloroplast genome sequence

Solanum berthaultii is one of the wild diploid Solanum species, which is an excellent resource in potato breeding owing to its resistance to several important pathogens. On the other hand, sexual hybridization between S. berthaultii and S. tuberosum (potato) is limited because of their sexual incompatibility. Therefore, cell fusion can be used to introgress various novel traits from this wild species into the cultivated potatoes. After cell fusion, it is crucial to identify fusion products with the aid of molecular markers. In this study, the chloroplast genome sequence of S. berthaultii obtained by next-generation sequencing technology was described and compared with those of five other Solanum species to develop S. berthaultii specific markers. A total sequence length of the chloroplast genome is 155,533 bp. The structural organization of the chloroplast genome is similar to those of the five other Solanum species. Phylogenic analysis with 25 other Solanaceae species revealed that S. berthaultii is most closely located with S. tuberosum. Additional comparison of the chloroplast genome sequence with those of the five Solanum species revealed 25 SNPs specific to S. berthaultii. Based on these SNPs, six PCR-based markers for differentiating S. berthaultii from other Solanum species were developed. These markers will facilitate the selection of fusion products and accelerate potato breeding using S. berthaultii.


Introduction
Potato (Solanum tuberosum L.) is one of the most important crops in the world. Its production has been jeopardized seriously by diverse pathogens, particularly the oomycete pathogen, Phytophthora infestans, the causal agent of late blight (Rauscher et al. 2006). Therefore, potato breeding has focused on achieving high level resistance to late blight. Moreover, several resistant genes have been introgressed to improve the resistance to late blight from wild Solanum species into cultivated potatoes in potato breeding.
S. berthaultii originating from Bolivia, South America is a wild diploid species and a relative of potato (Pavek and Corsini 2001;Spooner and Castillo 1997). The species has been known as one of the important resources for resistance to several important pathogens such as Phytophthora infestans, potato virus Y etc. in potato breeding (Ewing et al. 2000;Nouri-Ellouz et al. 2016;Park et al. 2009;Rauscher et al. 2010;Tan et al. 2010). On the other hand, sexual incompatibility caused by the different ploidy levels of the genome and endosperm balance number (EBN) between this wild species and potato have restricted the utilization of this wild species for improvements during classical breeding processes (Cho et al. 1997;Oritz and Ehlenfeldt 1992). To overcome this sexual barrier for interspecific gene transfer, therefore, protoplast fusion has been attempted with the two different species for crop improvement (Bidani et al. 2007;Nouri-Ellouz et al. 2016). In potato breeding, this technique has also been applied and succeeded in producing interspecific somatic hybrids such as S. tuberosum (+) S. chaconese (Chen et al. 2013), S. tuberosum (+) S. brevidens (Barsby et al. 1984), S. tuberosum (+) S. nigrum (Binding et al. 1982), S. tuberosum (+) S. phureja (Putie et al. 1986) and S. tuberosum (+) S. commersonii . When somatic hybridization is performed successfully, molecular markers can be used for the identification and verification of the somatic hybrids to confirm the hybridity of both nuclear and organelle DNA ). This process is essential due to the fact that a large amount of nuclear and organelle DNA can be transferred between two sexually incompatible species (Guo et al. 2004).
The chloroplast, a photosynthetic intracellular organelle has a circular double-stranded DNA molecule on its own genome. Most chloroplast genomes contain 110-130 genes encoding up to 80 unique proteins, approximately 30 tRNAs and 4 rRNAs. The angiosperm chloroplast is usually 1151 65 kb in size and has a quadripartite organization comprised of two 12-75 kb inverted repeats (IR) separating the 80~90 kb large single copy (LSC) and 16~27 kb small single copy (SSC) regions (Yurina and Odintosova 1998). As shown in Table 1, several complete chloroplast genome sequences of Solanum species including S. tuberosum (KM489056 and NC008096), S. commersonii (KM489055), S. bulbocastanum (NC007943), and S. nigrum (KM489054) have previously been reported Chung et al. 2006;Daniell et al. 2006). Their chloroplast genomes are highly conserved in size, structure and gene organization including the presence of two copies of IRs separated by LSC and SSC regions as previously known (Palmer 1991;Raubeson and Jansen 2005;Saski et al. 2005;Sugiura et al. 1998). Nevertheless, diversification of chloroplast genome sequences derived from the comparison of complete chloroplast genome sequences among plant species provides sufficient information (Cho et al. 2015). In the chloroplast genomes of many plants, a number of mutations such as single nucleotide polymorphism (SNPs), and insertions/deletions (InDels) and structural changes such as inversions, and rearrangements of gene order have been reported (Calsa Junior et al. 2004;Jheng et al. 2012;Saski et al. 2005).
Previously, the chloroplast genome sequence of S. berthaultii was completed using next-generation sequencing technology and shortly announced (Park 2017). In this study, we described the result of the sequence in detail, compared it with those of other Solanaceae species, and developed specific markers for S. berthaultii.

Chloroplast Genome Sequencing
Sequencing was conducted using the Illumina Hiseq2000 (Illumina, SanDiego, CA, USA) platform at Macrogen (Macrogen, Seoul, South Korea) with the total genomic DNA of S. berthaultii (SB1-1). The chloroplast genome sequence was obtained by de novo assembly via the Phyzen bioinformatics pipeline (http://phyzen.com). A CLC assembler (ver. 4.06 beta, CLC Inc, Rarhus, Denmark) was used for de novo assembly. From the overall reads, reads with raw scores of 20 or less were discarded using the CLC-quality trim tool and the parameters of a minimum (300~1,000 bp) autonomously controlled overlap size were applied. From the total contigs, the principal contigs representing the chloroplast genome were retrieved by Nucmer (Kurtz et al. 2004) using the chloroplast genome sequence of S. tuberosum (KM489056) as the reference sequence ).
The results from BLASTZ analysis with the reference sequence arranged the representative chloroplast contigs in order (Schwartz et al. 2003) and they were connected to a single draft sequence by joining the overlapping terminal sequences followed by manual editing through a comparison with the chloroplast genome sequence of S. tuberosum (KM489056) reported by . Gene annotation was conducted using DOGMA (Wyman et al. 2004) and the circular map of the S. berthaultii chloroplast genome was generated using the software OrganellarGenomeDRAW (http://ogdraw.mpimp-golm.mpg.de) (Lohse et al. 2013).

Comparison of Chloroplast Genome Sequences
The chloroplast genome sequence of S. berthaultii identified in this study was compared with those of 25 Solanaceae species including eight Solanum species, S. tuberosum (DQ386163), S. commersonii (KM489054), S. bulbocastanum (NC007943), S. lycopersicum (DQ347959), S. pimpinellifolium (NC026882), S. peruvianum (KP117026), S. nigrum (KM 489055), and S. melongena (KU682719) obtained from the National Center for Biotechnology Information (NCBI). Sequence comparison and phylogenetic analysis were conducted using the chloroplast coding sequences of eight species by MEGA 6.0 (Tamura et al. 2013) and by a maximum likelihood and a maximum parsimony methods under PAUP* v4.0b10 (Swofford 2001), respectively. A total of 1,000 bootstap replicates and a 50% cutoff value for the bootstrap consensus tree were applied for the analysis. Multiple alignment was also performed using the complete chloroplast genome sequences of six species by ClustalW2 in EMBL (https://www.ebi.ac.uk/Tools/msa/clustalw2).

Results and Discussion
Complete Chloroplast Genome Sequence of S. berthaultii The complete chloroplast genome sequencing of S. berthaultii was performed using NGS (next generation sequencing) technology. A library produced by the Illumina PE standard protocol generated 2,506,011,690 bp sequences in total and their average read length was 348 bp. Three representative contigs were identified using the de novo assembly of paired end sequences of the entire genome. They covered the entire chloroplast genome sequence of S. tuberosum (KM489056) reported by  and arranged in order based on the results of BLASTZ analysis (Schwartz et al. 2003) (Fig. 1). The putative assembly errors curated by mapping 1,699.85x raw reads onto the final assembly, and PCR products of several regions including border sequences of a pair of inverted repeats (IRs), a small single copy region (SSC) and a large single copy region (LSC) validated by BigDye Terminator Cycle Sequencing with ABI3730 automated capillary sequencer finalized the completion of the chloroplast genome sequence. As a part of the results has shortly been announced (Park 2017), the size of the complete chloroplast genome sequence of S. berthaultii (GenBank accession no. KY419708) is 155,533 bp. It has the typical quadripartite structure including 25,593 bp IRs separated by SSC of 18,372 bp and LSC of 85,975 bp with a circular double stranded DNA molecule as usual in most plant plastids. The total length is slightly longer than those of other Solanum species (Table 1). The chloroplast genome of S. berthaultii contains 137 unique genes and 11 of them are duplicated in the IRs (Fig. 2). Fourty-eight tRNAs and eight rRNAs exist and nine and four of them are duplicated and inversely oriented in the IRs (Fig. 2 and Table 1). 59.2% coding region of the genome was identified and an   (Table 1). As reported by  and , the total length, the GC content, and the total number of predicted genes, tRNA and rRNA of the S. berthaultii chloroplast genome are highly conserved with those of other Solanum species.
Phylogenetic and Comparative Analysis of the Chloroplast Genome Sequences As shown in Figure 3, the phylogenetic relationship was analyzed using chloroplast coding sequence of S. berthaultii and those of the 25 Solanaceae species. The maximum parsimony and maximum likelihood analyses produced the same topology and most nodes of the phylogenetic tree were strongly supported by the high bootstrap values. The results showed that S. berthaultii belonged to the same clade in Solanum species as expected and S. berthaultii formed the sister of S. tuberosum. Interestingly, S. berthaultii was the closest species to S. tuberosum even though it was S. commersonii . The complete sequence of the S. berthaultii chloroplast genome was compared with those of the five other Solanum species including two different genotypes of S. tuberosum to develop molecular markers for the selection of S. berthaultii cytoplasm as a reasonable number of InDels or SNPs was previously detected by the complete alignment of the chloroplast genome sequences including both the coding and non-coding regions of the Solanum species (Chung et al., 2006;. A result of multiple alignment with six species generated by ClustalW2 in EMBL (https:// www.ebi.ac.uk/Tools/msa/clustalw2) showed that overall 39 and 1,788 regions for InDels and SNPs existed among the six Solanum species, respectively.

Discovery and Verification of Specific Molecular Markers for Distinguishing S. berthaultii from Potato and Other Wild Solanum Species
The multiple alignment of the chloroplast genome sequences of six different Solanum species revealed a lot of InDels and SNPs among the species. On the other hand, the InDels and SNPs were caused mostly by the sequences of S. nigrum or S. bulbocastanum (detail data not shown). This result was supported by the fact that those two species were farther phylogenetically from S. berthaultii than S. commersonii and S. tuberosum as shown in the result of phylogenetic tree (Fig. 3). Therefore, the criteria to search for the InDels or SNPs in which the sequences should be specific to S.   berthaultii, but mostly conserved in the five other Solanum species.
Only two of the 39 InDels detected were specific to S. berthaultii, but PCR-based molecular markers could not be developed using InDel specific primers. One base pair in the sequence of S. berthaultii was different from those of S. tuberosum and S. commersonii, 19 base pairs from that of S. bulbocastanum and six base pairs from that of S. nigrum on the first InDel region. Two base pairs in the sequence of S. berthaultii were different from those of S. tuberosum and S. commersonii, five base pairs from that of S. bulbocastanum and 438 base pairs from that of S. nigrum on the second InDel region. Therefore, this study focused on the SNPs identified among six species to develop PCR-based markers. A total of 25 of the 1,788 SNPs detected were specific to S. berthaultii. Five SNPs including four consecutive base pairs existed on the inverted repeat region. The regions on which the SNPs existed were distributed almost equally in both coding regions and noncoding regions throughout the entire chloroplast genome sequence although the number of InDels in the non-coding regions was reported to be detected much more than that in the coding regions of the chloroplast genome of Solanum species Chung et al. 2006).
Characterization of the chlorotype using the molecular markers is important for not only potato breeding, but also evolutionary studies in Solanum species (Bohs and Olmstead 1997;Hosaka and Sanetomo 2012). In previous studies, a random distribution of the chloroplast genome from a certain genotype during in vitro plant regeneration and somatic fusion was commonly observed in plant species including Solanum species, even though a high frequency of recombination occurred in the mitochondrial genome (Chen et al. 2013;Lössl et al. 2000;Mohapatra et al. 1998;Smyda-Dajmund et al. 2016;Xiang et al. 2004). Overall, the results suggest that the SNP-derived CAPS markers developed in this study can be used to discriminate S. berthaultii reliably from other Solanum species, to select the proper chlorotype from the fusion products between S. berthaultii and Solanum species and to facilitate potato breeding using S. berthaultii.