Plant-specific primers for the amplification of the nrDNA ITS region in fungus-associated Pulsatilla species

Yan-Lin Sun, Wan-Geun Park, Hyun-Kyung Oh and Soon-Kwan Hong* School of Life Sciences, Ludong University, Yantai, Shandong, 264-025, China. Department of Forest Resources, Kangwon National University, Chuncheon, 200-701, Korea. National Institute of Biological Resources, Environmental Research Complex, Gyoungseo-dong, Seo-gu, Incheon, 404708, Korea. Department of Bio-Health Technology, Kangwon National University, Chuncheon, 200-701, Korea. Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon, 200-701, Korea.


INTRODUCTION
The internal transcribed spacer (ITS) region contains two variable non-coding regions that are nested within the ribosomal DNA (rDNA) repeat between the highly conserved small subunit, 5.8S, and large subunit rRNA genes (Baldwin et al., 1995).Several features make it the most frequently utilized and convenient target region for molecular identification of plants, fungi, and animals (Álvarez and Wendel, 2003;Kress et al., 2005).The advantages of using the ITS region for identification of plant and fungus species are that (a) the entire ITS region is generally between 600 and 800 bp, that makes efficient amplification and sequencing easy; (b) the primers for amplifying the ITS region shared high universality, e.g. the earliest ITS primers, ITS1, ITS4, and ITS5 have gained a wide acceptance for work with fungal and plant ITS region (White et al., 1990); (c) the multicopy nature of the rDNA repeat makes the ITS region easy to amplify from small, dilute, or highly degraded DNA samples; (d) the ITS region has high species discrimination, that has been much demonstrated especially when used as a region for the barcoding and authentication of herbal medicinal materials (Álvarez and Wendel, 2003;Kress et al., 2005;Karehed et al., 2008).However, due to a very broad range of primer universality, currently available primers could readily amplify the ITS region from both plant and fungal DNAs.This disadvantage affects further progress of the ITS region in species phylogenetic studies.
In our laboratory work, this situation of false-positive PCR amplification has reached an unignorable level, e.g. using universal primers could amplify the ITS regions from not only plant DNA but also fungal DNA in some fungus-associated plant species, including Dracocephalum argunense, Scutellaria baicalensis, and Toona sinensis (data not published).In many natural situations, fungal contamination of plant materials may be inevitable, thus specific or preferential amplification of plant DNA would be desirable.Pulsatilla cernua (Thunb., Ranunculaceae) is a perennial herb distributed in Northeastern China, Korea, and Japan (Huang et al., 2002).It is a common early spring flower in China, but is an endangered plant in Japan, because of a rapid decline of populations in the last two decades (Naito and Nakagoshi, 1994).The dried roots of P. cernua have been widely used as traditional Chinese medicine (TCM) for thousands of years in China, mainly for the treatment of amoebic and bacterial dysentery (Xu et al., 2010).P. cernua is known for containing a large number of compounds, including triterpenoidal saponins, phytosterone and anthocyanins (Xu et al., 2011).Modern pharmacological studies demonstrated that P. cernua extracts had a number of effects, such as hypoglycaemic, antitumour, cognition enhancing, neuroprotective, cytotoxic and antiendotoxin activities (Sparg et al., 2004;Han et al., 2007;Seo et al., 2010).Another Pulsatilla species, Pulsatilla tongkangensis Y.N. Lee & T.C. Lee (Ranunculaceae), a perennial herb was discovered naturally growing on the cliffs of Tongkang, Kangwon province, Korea and endemic to Korea, thus was announced as a new Pulsatilla spp., P. tongkangensis (Lee, 2000;Lee et al., 2010;Kim et al., 2010).From the morphological characters, P. tongkangensis has a shorter length of stem, and flower stalk, compared to other Pulsatilla spp.As known, the flower stalk of most of Pulsatilla spp.bends during their anthesis, like P. cernua (Huang et al., 2002), however, the movement of the floral stalk during anthesis could not been found in P. tongkangensis.In Korea, there are six main Pulsatilla spp.naturally growing in the welldrained gravel areas or sandy soils or even cavity of calcified rocks, including P. cernua, Pulsatilla davurica, Pulsatilla koreana, Pulsatilla nivalis, Pulsatilla chinensis, and P. tongkangensis (Lee et al., 2004).Because of the sharing of similar morphological characteristics, traditional identification methods have not been able to achieve clear discrimination and effective use of the Pulsatilla spp., molecular identification based on analyses of DNA sequence diversity becomes more reliable alternative means.
In this study, we have successfully amplified the ITS region from P. cernua using universal primers, but with poor plant-specificity.The ITS region could not be effectively amplified using universal primers from natural P. tongkangensis materials.The reason of inefficient amplification of plant ITS region was the come down to the severe fungal contamination and super high primer universality; the reason of unsuccessfully amplification was believed to owe to the mismatch of the ITS primers with P. tongkangensis DNA sequence.To achieve the effective amplification of ITS region from P. tongkangensis, we have designed and tested one new primer set (ITS1-F2/ITS4-R) that in combination specifically amplify the ITS region of plants from funguscontaminated plant materials.This plant-specificity of newly designed primer set was also suitably applied to the ITS region amplification from P. cernua.In combination with universal primers, the newly designed primers could also achieve the preferential ITS amplification from plants.Our focus on plant-specific ITS primers could help more accurately, rapidly amplification of the ITS region from plant materials, even some fungusassociated plant materials.In addition, yet little is known, the ITS region sequences of the new species, P. tongkangensis, that furthermore makes against the understanding of the phylogeny between P. tongkangensis and other Pulsatilla spp.The ITS region sequences of P. tongkangensis presented here is the first time to successfully amplify and sequence.Thus, this study also provides more sequence sources to compare to further understand the phylogenetic relationships of the genus Pulsatilla.

DNA extraction and PCR amplification
DNA extractions were performed by using the modified cetyltrimethylammonium bromide (CTAB) method described by Doyle and Doyle (1987).PCR was performed using a Gene Amp 9700 PCR system (Applied Biosystems Inc. ABI, Warrington, Cheshire, UK) in 20 to 25 µl volumes with the following reaction components: 1 µl template DNA (~1 to 100 ng), 10 × Ex Taq Buffer (TaKaRa, TAKARA BIO INC., Japan), 200 µmol/l each dNTP, 0.1 µmol/l each primer, and 0.1 µl TaKaRa Ex Taq (5 units/µl, TaKaRa, TAKARA BIO INC., Japan).PCR amplification conditions followed an initial denaturation stem of 95°C for 5 min, 35 amplification cycles of denaturation at 95°C for 1 min, annealing at 50°C for 1 min, and a final extension step at 72°C for 1.5 min.After the 35 cycles were completed, the samples were incubated for an additional 7 min at 72°C.Two universal ITS primers, ITS5 and ITS4 (White et al., 1990), were used to combine with new designed ITS primers, ITS1-F2 and ITS4-R, for more accurately and preferentially amplification from fungus-contaminated plant materials.The accurate location of the newly designed ITS primer is as shown in Figure 1.The amplification products were checked by electrophoresis through 1.0% agarose gel, and then purified before DNA sequence analysis using a QIAquick PCR Purification Kit (QIAGEN, Korea) or Gel Purification Kit (QIAGEN, Korea) according to the manufacturer's instructions.

Plant-specific ITS primer design
Two plant-specific primers for the ITS region, ITS1-F2 and ITS4-R were designed, located in the 3' end of the 18S rDNA and the 5' end of the 28S rRNA gene, respectively (Figure 1).The sequences used for comparison were obtained from NCBI GenBank database, with Accession Numbers of GU732648 ~ GU732651, JF422893, HQ829829, HQ829821, HQ735289, FJ639908, JF422890 ~ JF422892 for different Pulsatilla spp.and GU732647, HQ898650, AB120213, AM267279 for different fungus species parasitic in Pulsatilla spp.
The 3' end of 18S rDNA sequences shared relatively high homology between Pulsatilla spp.and parasitic fungus species, while the 5' end of 28S rDNA sequences showed some informative nucleotide sites, easy to design plant-specific ITS primer.

Sequence editing and alignment
For editing and assembly of the complementary strands of the sequencing results, the software program DNAMAN version 6.0 (Lynnon Biosoft Corporation, USA, www.lynon.com)was used.Analogue of our sequences and nucleotide sequence comparisons were detected with Basic Local Alignment Search Tool (BLAST) network services against databases (http://www.ncbi.nlm.nih.gov/).To detect single nucleotide polymorphisms, the multiple sequence alignments of ITS region (ITS1, 5.8S rRNA gene and ITS2) of all the16 Pulsatilla spp.and 6 parasitic fungus species were respectively performed also using DNAMAN version 6.0 software.

Phylogenetic analysis
The phylogenetic relationships among the Pulsatilla spp. was estimated after the construction of a phylogram based on multiple sequence alignment of ribosomal DNA ITS sequence with the DNAMAN version 6.0 software (Lynnon Biosoft Corporation, USA, www.lynon.com).The relevant region lengths (ITS1, 5.8S rRNA gene and ITS2) of each sequence were calculated.Genetic distance (GD) was obtained with the help of MEGA software and mean GD of the intraspecific distance was calculated by the sum of individual GD divided by the number of samples.

RESULTS AND DISCUSSION
Using ITS region as a DNA marker began with discriminating some closely-related fungus species and understanding their phylogenetics (White et al., 1990).As the development of plant systematic and identification, the discrimination through molecular identification approach has gradually replaced the traditional plant discrimination.And the ITS region, due to its high primer universality, has become the single most frequently utilized DNA region in plants, fungi, and even animals (Hershkovitz et al., 1999).However, high primer universality also has a disadvantage that these primers simplify a wide range of plant targets and work well to analyze DNA isolated from individual organisms, but do not exclude effectively the fungus sequences in mixed, phytosphere DNA extracts of fungus-associated plants.
To resolve this problem, the plant-specific primers should be subsequently designed for analyses of plant ITS sequences (Zhang et al., 1997;Cullings and Vogler, 1998).However, due to the nucleotide variations occurring among not only different plant species but also different fungus species, newly designed primers able to be specific to plants were intended to be specific for certain plant species and specific-associated fungus species.

Plant-specific ITS primer design
In this study, the Pulsatilla spp.specific primers of ITS region were newly designed, named by ITS1-F2 and ITS4-R as forward and reverse primer, respectively.Due to relatively high conservation of the 18S and 28S rRNA region, it makes it convenient to design universal primer for the amplification of the ITS region, but also hard to select to amplify certain organisms from potential other species-contaminated materials.When comparing all existing Pulsatilla spp.and fungus species parasitic in Pulsatilla spp., the accurate sequence alignment was too hard to compare the matches between plant and fungal DNA sequence.Thus, respective sequence alignment of plant and fungi could only be performed in this study.As mismatches at the 3' end of primer are often most critical for efficient amplification (Sommer and Tautz, 1989;Kwok et al., 1990), we tried to find mismatches between plant and fungus sequences at the 3' end of the primer.However, the 18S rRNA shares very highly conserved sequences among the Pulsatilla spp.and their parasitic fungus species, that the newly designed ITS1-F2 primer might match both plant and fungal DNAs well (Figure 1).For the reverse primer, it is easier to find some sequence part that matches plant DNA but not fungal DNA, because the fungal ITS sequences among the 5' end of the 28S rRNA gene were variable to the ITS sequences of plants (Figure 1).Therefore, the newly designed primer, ITS4-R was very specific and sensitive to Pulsatilla spp.Besides Pulsatilla spp., the high plant-specificity of the ITS4-R primer was also investigated and tested on other plant species with potential fungal contamination in our laboratory work, e.g.Scutellaria baicalensis Georgi had shown high plant-specificity of the ITS4-R/ITS1-F2 primer set (data not published).

PCR amplification
The amplification ability of four primer pairs including ITS4/ITS5, ITS1-F2/ITS4-R, ITS1-F2/ITS4, and ITS5/ITS4-R, were tested in this study (Figure 2).The amplification results were very different from both P. cernua and P. tongkangensis, and primer pairs.The P. cernua DNAs could amplify efficiently with all of the primer pairs, but using ITS4/ITS5 and ITS1-F2/ ITS4 primer pairs produced two bands.After purifying and sequencing these both PCR products, it was known that the bigger size product was plant ITS region and the smaller size product was the fungal ITS region.Using ITS1-F2/ITS4-R and ITS5/ITS4-R primer pairs, it could preferentially amplify a single clear band from the funguscontaminated P. cernua DNAs.Compared with the size of PCR product, it was trended to be plant ITS region, and the suggestion was strongly supported by our sequencing results.It was said that in combination of ITS4-R primer, the plant DNA isolated from natural P. cernua materials was preferentially amplified to the apparent, exclusion of fungal DNA (Figure 2).This result suggested that ITS4-R was specific to P. cernua DNA but not fungal DNA, while other three primers including ITS1-F2, ITS4, and ITS5 were not specific neither to P. cernua DNA nor fungal DNA.For the new species, P. tongkangensis, the universal primer set, ITS4/ITS5 did not work when used to amplify the ITS region, resulting in either no product or wrong amplified product (Figure 2).This result indicated that both or at less one of both universal primers mismatched the P. tongkangensis ITS region.So, the occurrence of this result absolutely puzzled us at the beginning, because there was no any sequence sources of P. tongkangensis published in GenBank database of NCBI, that limits the free comparison of DNA sequences of P. tongkangensis with those from other plant species.Luckily, the newly designed primers, ITS1-F2 and ITS4-R with the original design intention of Pulsatilla spp.specific ITS primers, could amplify the ITS region from funguscontaminated P. tongkangensis materials, and the amplification ability was not low.Using both newly designed primers, ITS1-F2 and ITS4-R, not only effectively amplified the ITS region from P. tongkangensis, but showed very high plant-specification.However, when combined with one universal primer, ITS4 or ITS5, the amplification ability was largely decreased: in combination with the universal primer ITS4, the reactions could successfully amplify, but the plant-specification was low; in combination with the universal primer ITS5, the reactions could not produce any accurate amplification from neither plants nor fungi.These results suggested that ITS1-F2 might well match the ITS regions of both P. tongkangensis and parasitic fungi but not ITS4-R, and the universal primer ITS4 could match P. tongkangensis DNA, but not another universal primer ITS5.Due to the primer location of ITS1-F2 existing in the downstream of that of ITS5, the accurate, relevant primer binding site of ITS5 in P. tongkangensis DNA sequence could not be checked in the present work, but the hypothesis that universal primer ITS5 did not match P. tongkangensis would be proved using an upstream primer of ITS5 in the future.

Single nucleotide polymorphisms between P. cernua and P. tongkangensis
To further understand the phylogeny between P. cernua and P. tongkangensis, the single nucleotide polymorphisms were analyzed in this study.In the total ITS region, there were 29 nucleotide substitutions or indels obtained in all, among which 9 nucleotide variable sites were located in the ITS1 region and 20 sites in the ITS2 region (Table 2).The single nucleotide polymorphisms occurred not only between P. cernua and P. tongkangensis, but among accessions of same species.Among all the variable nucleotide sites, five ones at position 9, 74, 418, 472, and 537 bp were species-specific, occurring completely between P. cernua and P. tongkangensis but not among accessions of each species (Table 2).To understand whether these variable sites were P. tongkangensis-specific, single nucleotide polymorphism were further analyzed combined with other Pulsatilla spp. at these nucleotide sites (Table 3).The detailed result was presented in the following part of phylogenetic relationship among the Pulsatilla spp.
Decently, accumulating evidence suggests that nucleotide variations might occur among ITS 1 The number means the nucleotide site number in the complete ITS1 and ITS2 region of Pulsatilla species.R is the combination of A and G; Y is the combination of T and C; .means nucleotide site indel.
sequences even in one organism, because this kind of organisms are hybrids, either diploid or polyploids, of disparate parents (Sang et al., 1995;Buckler et al., 1997).
For both Pulsatilla spp., some nucleotide variations showed accession-specific, that might express the hybridization result.Although there was no report indicating that P. tongkangensis could be hybridized with P. cernua, a hybridization behavior between P. tongkangensis and P. koreana, and was suggested based on randomly amplified polymorphic DNA (Lee et al., 2010).Due to initially growing in a very isolated area in Tongkang, Korea, P. tongkangensis was recorded as a new species and identified based on leaf morphology, the orientation of flower buds before anthesis, and flower colors (Lee, 2000;Lee and Kim, 2006).However, P. cernua, as one of the mostly common Pulsatilla spp. in Korea like P. koreana, made natural hybridization possible between these two species.The mostly apparent variable site typical of hybridization was position 70 bp, that both P. cernua accessions HN1 and HN2 showed C at this site, while most P. tongkangensis accessions showed nucleotide substitution with T, excluding accession PH4, PH5, PH10, and PH13 (Table 2).In particular, PH5 showed combination with T and C at position 70 bp, further suggesting that P. tongkangensis could be hybridized with other Pulsatilla spp.In addition, position 2 and 3 bp might also represent the hybrid result, that P. cernua accession HN1 shared nucleotide indel at position 2 bp with most P. tongkangensis accession exclusion of PH1 and PH4, and HN2 shared the same nucleotide sequence with PH1 and PH4 at position 2 bp; on the contrary, HN2 shared nucleotide indel with PH1 and PH4 at position 3 bp, and HN1 showed the same nucleotide sequence as other P. tongkangensis accessions (Table 2).
Besides with P. cernua and P. koeana, P. tongkangensis might be capable to hybridize with other Pulsatilla spp., such as P. davurica, P. nivalis, and P. chinensis growing in the overlapping geographic areas in Korea.The occurrence probability of hybridization made the understanding of accession-specific variable nucleotide sites more easily, e.g.position 580 and 589 bp in PH6 showed one nucleotide insertion, position 594 and 599 bp in PH8 showed one nucleotide substitution, and position 609 bp in PH5 had one nucleotide insertion (Table 2).

Phylogenetic relationship among the Pulsatilla spp.
Despite the fact that the ITS region sequence was available in many Pulsatilla spp.including Pulsatilla violacea, P. cernua, P. chinensis, Pulsatilla dahurica, Pulsatilla halleri, Pulsatilla rabra, Pulsatilla turczaninovii, and Pulsatilla albana, no ITS sequence source but any other sequence sources of P. tongkangensis were found in NCBI GenBank database until were submitted in the early October in 2011.In general, sequences from samples difficult to amplify and sequence were scarcely few.For P. tongkangensis, besides this reason, its habitat endemic to Korea also led to the scarcity of sequence sources.According to the DNA alignment result, there were 59 nucleotide variable sites occurring in all among the 571 bp common length of the ITS region of Pulsatilla spp.(data not shown).Not like assumed, nucleotide variations did not mainly limit between P. tongkangensis and other Pulsatilla spp., on the contrary, some Pulsatilla spp.showed identical nucleotide sequences to P. tongkangensis but not to the common species, P. cernua.At the four P. tongkangensis-specific variable sites, this  situation was also found, e.g.P. violacea, P. chinensis, P. dahurica, P. rubra, P. turczaninovii, and P. albana shared the same nucleotide sequence at site 74, 472, and 537 bp (Table 2).One hybrid species by P. cernua and P. dahurica (HQ829821) showed clear homogenization to that of either parental species due to the concerted evolution in these four nucleotide variable sites, with site 74 and 472 bp from both parental species and site 537 bp from P. dahurica species.Although there was one P. dahurica sequence showing that site 537 bp is identical to P. cernua but not the P. cernua × P. dahurica hybrid species; the ITS region of this hybrid species showed high homogenization level.The new species, P. tongkangensis had only one specific variation at site 418 bp.
In the phylogenetic tree constructed by all the ITS region sequences, both P. cernua accessions formed a monophyletic group, sharing 99% of homology with each other, while P. tongkangensis accessions showed three one-or multi-level groups, parallel to the P. cernua monophyletic group (Figure 3).In the phylogenetic tree constructed by the ITS region of all the Pulsatilla spp., no (99.60%),followed by P. rubra and P. halleri (99.50%,Table 4).
The homology rate between some different species was even higher than that among different accessions of the same species, suggesting that nucleotide variation among all the Pulsatilla spp. was not only geographical origin-related but also species-related.Aligning DNA sequences of all the Pulsatilla spp., the identity rate among them reached a relatively high level of 97% (Figure 4).

Conclusion
Fungus contamination always puzzles the ITS amplification from natural plant materials, particularly some medicinal plant species.Two newly designed primers specific for Pulsatilla spp.presented here is anticipated to serve as the basis for a wide ranging system to analyze plant materials, particularly in association with fungi.In addition, these new primers sharing high universality provide us more combinations of primer sets for the amplification, and could also supplement some cases that are difficult to successfully amplify using universal primers, such as P. tongkangensis investigated in this study.By using multiple primer sets, we expect to maintain a wide range of universality and a high degree of comparability between PCR reactions.And these multiply primer sets are being attempted for plant-specific ITS amplification in other plant species materials in our laboratory.Two newly designed primers were developed with an emphasis on discrimination between plant and fungal sequences and should be particularly useful for studies of plant materials where samples also contain high levels of background fungal DNA.

Figure 1 .
Figure 1.Accurate location of the newly designed ITS primers, ITS1-F2 and ITS4-R.ITS1-F2 and ITS4-R are targeted in the 3' end of 18S rRNA and 5' end of 28S rRNA gene, respectively.All primer sequences are listed in the standard 5' to 3' direction.

Figure 3 .
Figure 3. Phylogenetic tree constructed using the ITS region sequences of two P. cernua accessions and fourteen P. tongkangensis accessions.

Table 1 .
Lee plants were conserved by Prof. Wan-Geun Park, and cultured in the green house of Department of Forest Resources, Kangwon National University.There are fourteen accessions of P. tongkangensis in all.Their specimen vouchers and NCBI GenBank accession numbers of the ITS region are listed in Table1.Specimen vouchers and NCBI accession numbers of plant and fungus materials investigated in this study.

Table 2 .
Comparisons of nucleotide variable sites occurring between P. cernua and P. tongkangensis investigated in this study.

Table 3 .
Comparisons of four nucleotide variable sites occurring only between P. cernua and P. tongkangensis in this study, with other existing Pulsatilla species.

Table 4 .
Genetic distance (GD) and homology rate evaluated by pairwise percentage similarity of the ITS region of species of Pulsatilla species.
GD was presented below the axlewire; homology rate was presented above the axlewire.