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First published online March 7, 2008; 10.1104/pp.108.116137 Plant Physiology 147:216-227 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
The Central Role of a SNRK2 Kinase in Sulfur Deprivation Responses1,[W],[OA]Department of Plant Biology, Carnegie Institution, Stanford, California 94305 (D.G.-B., A.R.G.); Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803 (S.V.P.); and Department of Biological Sciences, Stanford University, Stanford, California 94305 (W.P.)
In the absence of sulfur (S), Chlamydomonas reinhardtii increases the abundance of several transcripts encoding proteins associated with S acquisition and assimilation, conserves S amino acids, and acclimates to suboptimal growth conditions. A positive regulator, SAC1 (for sulfur acclimation protein 1), and a negative regulator, SAC3, were shown to participate in the control of these processes. In this study, we investigated two allelic mutants (ars11 and ars44) affected in a gene encoding a SNRK2 (for SNF1-related protein kinase 2) kinase designated SNRK2.1. Like the sac1 mutant, both snrk2.1 mutants were deficient in the expression of S-responsive genes. Furthermore, the mutant cells bleached more rapidly than wild-type cells during S deprivation, although the phenotypes of ars11 and ars44 were not identical: ars11 exhibited a more severe phenotype than either ars44 or sac1. The phenotypic differences between the ars11 and ars44 mutants reflected distinct alterations of SNRK2.1 mRNA splicing caused by insertion of the marker gene. The ars11 phenotype could be rescued by complementation with SNRK2.1 cDNA. In contrast to the nonepistatic relationship between SAC3 and SAC1, characterization of the sac3 ars11 double mutant showed that SNRK2.1 is epistatic to SAC3. These data reveal the crucial regulatory role of SNRK2.1 in the signaling cascade critical for eliciting S deprivation responses in Chlamydomonas. The phylogenetic relationships and structures of the eight members of the SNRK2 family in Chlamydomonas are discussed.
Sulfur (S) is an essential element present in proteins, lipids, carbohydrates, electron carriers, various metabolites (some involved in the detoxification of heavy metals and xenobiotics), and signaling molecules (Meister and Anderson, 1983  ; Gupta et al., 1990; Schultze et al., 1992; Marrs, 1996; Grossman and Takahashi, 2001). The preferred S source for most organisms is sulfate (SO42–), which can be limiting in the environment; limitations for SO42– may result in reduced quality and yield of seeds and cause stunted plant growth (Mahler and Maples, 1986, 1987; Warman and Sampson, 1994). Most organisms are unable to efficiently store S; therefore, they are dependent on a continuous supply of this nutrient.
When organisms become S limited, they exhibit a suite of responses that have been described as either specific or general. The specific responses to nutrient limitation are those associated with deprivation for a single nutrient and are often involved in scavenging or conserving that specific nutrient. For example, during S deprivation, Chlamydomonas reinhardtii synthesizes periplasmic arylsulfatases (ARS) that catalyze the hydrolysis of organic SO42– esters (de Hostos et al., 1989
Very little is known about regulatory mechanisms that control S nutrition in vascular plants and other photosynthetic eukaryotes. The generation of insertional mutants of Chlamydomonas has resulted in the identification of two specific regulators of S deprivation responses, SAC1 (sulfur acclimation protein 1) and SAC3. SAC1 plays a central role in controlling S deprivation responses (Davies et al., 1996
SAC1 has significant sequence similarity to the animal S transporters (Na+/SO42– transporters). Three additional genes that encode proteins with strong sequence similarity to mammalian Na+/SO42– transporters (SLT1–SLT3) have been identified on the Chlamydomonas genome. The physiological effects of the sac1 mutation strongly imply that SAC1 is a hierarchical regulator critical for the acclimation of cells to S deprivation and raise the possibility that transporter-like proteins might have evolved into sensor proteins that are critical for acclimation processes (Davies et al., 1996
The SAC3 S deprivation regulator of Chlamydomonas is a putative Ser/Thr kinase in the plant-specific SNF1-related protein kinase 2 (SNRK2) family. The sac3 mutants exhibit low-level constitutive ARS activity in S-replete medium, but like wild-type cells, they accumulate high levels of ARS following exposure of the cells to S deprivation. Other S-responsive genes (in addition to ARS) are also negatively regulated by SAC3 (Ravina et al., 2002
Recently, a collection of mutants that exhibit low ARS activity and that are potentially affected in S deprivation responses was generated by insertional mutagenesis (Pollock et al., 2005
Isolation of the ars Mutants
Insertional mutants (disrupted with the AphVIII marker gene) of Chlamydomonas were screened for abnormal levels of extracellular ARS activity after transferring cells to TAP-S medium (for Tris-acetate phosphate medium without S). Two kinds of mutants were identified: those with no or low ARS activity (ars– mutants) and those that exhibited more ARS activity than the parental strain (ars+ mutants). Approximately 30,000 transformants were screened, and from those 50 strains exhibited an ars– phenotype and two exhibited an ars+ phenotype (Pollock et al., 2005
An adaptor-mediated PCR technique (Padegirnas and Reichert, 1998
To determine whether the ars– phenotype was linked to the AphVIII insertion, the ars11 and ars44 mutants were crossed with the wild-type 21gr strain, which is sensitive to paromomycin. For both mutants, the paro+ progeny (approximately 30 of a total of 60 analyzed) cosegregated with the ars– phenotype, strongly supporting the conclusion suggested by the occurrence of two alleles with related phenotypes that the insertion was the cause of the mutant phenotype.
The ars11 and ars44 mutants exhibited no or low ARS activity when the cells were deprived of S, although the phenotypes of the strains were not identical. Like sac1, ars11 had no detectable ARS activity on agar plates, while ars44 exhibited reduced ARS activity; the ars44 strain accumulated substantial levels of ARS activity if the cells were maintained on TAP-S plates for a long enough period of time (Fig. 1A ). In liquid cultures, ars44 showed essentially no ARS activity after 24 h of S starvation (Fig. 1B), and even after several days of S starvation, very low-level activity was observed (data not shown).
Davies et al. (1996) showed that the sac1 strain is defective for the specific and general S deprivation responses. One feature of sac1 is its rapid loss of chlorophyll and death, relative to wild-type cells, following the imposition of S deprivation; this death response may be a consequence of the inability of the mutant to decrease the rate of photosynthetic electron flow through PSII (Davies et al., 1996). The ars11 and ars44 mutants also bleached (Fig. 1C) and died much more rapidly than wild-type cells when transferred to TAP-S medium, suggesting that, like sac1, these strains are aberrant for both the specific and general S deprivation responses. Interestingly, the ars11 strain bleached and died significantly more rapidly than either ars44 or sac1.
To further analyze the responses of the ars11 and ars44 strains to S deprivation, the levels of transcripts from several genes that were already known to be controlled by the S status of the medium were analyzed; these genes included ARS1 for the arylsulfatase (de Hostos et al., 1989
The cDNAs and Sites of Insertion in ars11 and ars44 There were no EST sequences to help identify the complete coding sequence (CDS) and intron-exon boundaries of the SNRK2.1 genomic sequence. Therefore, a 2,129-bp cDNA was amplified by reverse transcription (RT)-PCR from the parental strain RNA. Based on the sequence of the cDNA, the gene contains a 1,173-bp CDS with an 810-bp 3' untranslated region (UTR) and a 146-bp 5' UTR. Other cDNA sequences that could represent alternative splice forms of the SNRK2.1 transcript were also amplified (see below). The cDNA sequence was different from the predicted sequence of the ab initio model for the gene that was generated by the Joint Genome Institute (JGI; http://genome.jgi-psf.org/Chlre3/Chlre3.home.html). We recently updated this model to take into account cDNA sequence information. The genomic sequence corresponding to the cDNA was 5.46 kb in length, consisting of 11 exons and 10 introns (Fig. 3A ), encoding a predicted polypeptide of 390 amino acids.
Adaptor-mediated PCR was used to identify one of the genomic regions flanking the AphVIII marker gene for both ars11 and ars44 (the border close to the 3' end of AphVIII; Pollock et al., 2005 ). In ars11, the insertion was positioned within exon VII, whereas in ars44, the insertion was within intron 6 (Fig. 3B). To identify the flanking regions on both sides of the insertions, specific primer pairs were designed to anneal to the SNRK2.1 genomic sequence close to the site of insertion and to the marker gene. Amplifications using these primers and analysis of the flanking sequences showed that neither of the insertion events was accompanied by deletions or by a reorganization of the genome around the site of the insertion (Kindle, 1990). Using RT-PCR, we demonstrated that neither ars11 nor ars44 was completely lacking SNRK2.1 transcripts; however, the mRNA from SNRK2.1 in ars11 was missing the sequence encoded by exon VII, while ars44 retained a full-length SNRK2.1 transcript that was indistinguishable from that of the parental strain (Fig. 3C). In ars11, splicing resulted in a fusion of exon VI to exon VIII, which eliminated exon VII (the site of the insertion) from the processed transcript (Fig. 3C). Furthermore, the translated protein of the ars11 mutant would have a shift in the reading frame generated as a consequence of the fusion of exons VI to VIII. When SNRK2.1 transcript accumulation was determined using primers specific for the 3' UTR, no significant difference between the mutant and wild-type transcript levels was observed (data not shown). However, when SNRK2.1 transcript accumulation in ars11 was determined using primers specific for exons VI and VII, no transcript was detected. In contrast, the same primers did reveal the presence of a transcript with the proper fusion of exon VI and VII in ars44, although the transcript was reduced by approximately two orders of magnitude in this mutant strain (with the greatest reduction observed in cells exposed to 4 h of –S conditions) relative to parental cells (Fig. 4
). Thus, the ars44 strain is likely able to synthesize normal SNRK2.1 protein, but the level of this protein may be significantly lower than in parental cells because of the low-level accumulation of the mRNA. The molecular differences associated with mRNA processing are likely responsible for the different mutant phenotypes. Furthermore, the data demonstrate that splicing of intron 6 of SNRK2.1 can occur even if the intron is interrupted with the 1.7-kb PSAD::AphVIII marker gene.
Potential Alternative Splicing of SNRK2.1
As mentioned above, SNRK2.1 cDNA generation led to multiple cDNAs of different lengths. At least five different SNRK2.1 cDNAs were identified using the same set of specific primer pairs for RT-PCR (the RNA template was from D66 cells grown in TAP and TAP-S); these cDNAs were designated SNRK2.1 v1, v2, v3, v4, and v5, and the differences among them are shown in Supplemental Figure S1. SNRK2.1 v1 was the longest cDNA, encoding a polypeptide of 390 amino acids, and is the isoform discussed above (referred to as SNRK2.1). All of these potential transcripts encoded putative polypeptides that used the same start codon, with SNRK2.1 v1, v2, and v3 maintaining the same coding frame and stop codon. In contrast, the SNRK2.1 v4 and v5 reading frames changed as a result of the alternative splicing, which also generated a different stop codon. For SNRK2.1 v2 to v4, the potential alternative 5' splice sites were positioned inside a 54-bp GC-rich region of the transcript. The deduced SNRK2.1 v2 protein maintained the kinase catalytic domain and the end of the C-terminal domain, whereas v3 lacked 20 amino acids from the kinase domain and part of the C-terminal region. The SNRK2.1 v4 and v5 predicted polypeptides lacked the kinase domain and the entire C terminus. An alignment of all of the potential splice variants is shown in Supplemental Figure S2. None of the potential splice variants was associated with a specific culture condition (e.g. TAP compared with TAP-S), and the only variant reproducibly observed was SNRK2.1 v1; the others were less abundant and in some reactions were not detected. Finally, only in SNRK2.1 v1 did all of the splice junctions match the consensus junctions associated with Chlamydomonas genes (Silflow, 1998 To help establish if the small SNRK2.1 transcripts were potentially artifacts, we used specific primers to generate PCR products from a pGEM-T clone harboring the SNRK2.1 v1 cDNA. A multiband amplification pattern was observed, and at least one of the products had a gap in the sequence starting within the same GC-rich region that was identified as the 5' splice junction for putative splice variants. However, the products generated by PCR from the plasmid did not perfectly match any of the putative SNRK2.1 splice versions (data not shown). These observations raise the possibility that putative alternative splice forms can arise from aberrant cDNA synthesis and amplification in vitro, and not from in vivo splicing of precursor mRNA.
Four of the SNRK2.1 cDNA potential splice variants (v1, v2, v3, and v4) were introduced into the ars11 mutant strain. Each cDNA version was inserted between the PSAD promoter and the 3' UTR terminator and cloned into a vector containing the ble gene as a selectable marker. Fifty ble-resistant colonies for each splice variant were tested for ARS activity following exposure to TAP-S. Four colonies transformed with the SNRK2.1 v1 cDNA restored the wild-type ARS phenotype, based on ARS plate assays. An analysis of ARS activity in liquid for one of the potentially complemented mutant strains (tars11#34) showed that the strain was rescued for the ars– phenotype; it produced high levels of ARS when deprived of S (Fig. 1B), with no ARS activity detectable when grown in TAP (data not shown). No complemented colonies were obtained that harbored v2, v3, or v4 cDNA variants. Analysis of chlorosis of the tars11#34 strain in TAP-S also revealed restoration of the wild-type phenotype (Fig. 1C); transformants expressing ARS survived S deprivation for an extended period of time, like the D66 parental strain. Finally, the levels of ARS1, SLT1, and SULTR2 mRNAs measured in tars11#34 were similar to those of the parental strain (Fig. 2G). These results clearly demonstrated that the mutant phenotype can be complemented by the full-length SNRK2.1 gene.
SNRK2.1 is required for the activation of genes associated with S deprivation responses, while SAC3 acts as a negative regulator of at least some of these same genes (Davies et al., 1999
SNRK2.1 belongs to the plant-specific Ser/Thr protein kinase family SNRK2. Like other SNRK2 family members, SNRK2.1 has an N-terminal conserved catalytic domain similar to those of SNF1/AMP kinases and a short C-terminal regulatory domain that is not highly conserved. SAC3 (Davies et al., 1999
In this study, we identified SNRK2.1, a member of the plant-specific SNRK2 kinase family and the larger SNF1 superfamily, as a key regulator of the pathway that governs S deprivation responses in Chlamydomonas. Two independent mutant strains in which the SNRK2.1 gene was interrupted, ars11 and ars44, were identified. The insertions did not cause reorganization or deletions of the genomic region at the insertion site, and the mutant phenotypes were linked to the insertions. Even though the mutants had a sequence of 1.7 kb (AphVIII under the control of the PSAD promoter and the 3' UTR) integrated into the SNRK2.1 gene, the splicing machinery of Chlamydomonas was able to excise the inserted sequence and generate a mature mRNA; this mRNA was aberrant in the case of ars11. Insertion of the AphVIII marker gene into exon VII of SNRK2.1 in ars11 caused the loss of this exon in the final splice product and a change in the reading frame of the C-terminal region of the protein. These modifications of the SNRK2.1 protein are likely the reason for the severe mutant phenotype (the lesion probably represents a null mutation). In contrast, in ars44, the marker gene is integrated into intron 6. This interrupted intron (with the inserted marker DNA) was spliced out of the nascent transcript, generating a mature transcript that appeared to be identical to the transcript in the parental strain, although the level of accumulation of this mature transcript was more than 10 times lower than in the parental strain (Fig. 4). Therefore, the leaky phenotype of ars44 is probably a consequence of inefficient splicing of intron 6, which in turn could generate less protein. Interestingly, in spite of a clear difference in ARS activity between ars11 and ars44 mutants based on agar plate assays (Fig. 1A), both strains showed no ARS activity when they were transferred to liquid TAP-S medium for 24 h (Fig. 1B); there was little or no ARS activity measured in either of the mutants, even after several days of S starvation (data not shown). Several hypotheses might explain these findings. (1) There may be a difference in some crucial condition (e.g. oxygen availability) that results in elevated induction/activation of ARS activity on solid medium. This idea is supported by the finding that cells growing under the agar, at lower oxygen levels, exhibit higher ARS activity than cells growing on top of the agar (data not shown). (2) The splicing machinery may be somewhat different under different growth conditions, which might change the efficiency at which some transcripts are spliced. (3) ARS stability could be lower in cells growing in a liquid environment. Further experimentation should distinguish these possibilities.
The analysis of multiple potential splice variants of the SNRK2.1 transcript has raised critical issues concerning the in vivo synthesis of these variants and whether the procedure for cDNA synthesis could lead to their artifactual generation. The likelihood of an artifact generated during the synthesis of the cDNA is supported by the finding that the sequences of the splice junction sites for the potential variants identified differ markedly from the Chlamydomonas consensus splice junction sequences (Silflow, 1998
The SNRK2, SNRK1, and SNRK3 kinase families are plant specific and belong to the SNF1 superfamily. SNRK2 kinases constitute a large protein family in plants with an N-terminal conserved catalytic domain and a short regulatory C-terminal region. The regulatory region is not highly conserved but usually has a characteristic stretch of the acidic amino acids Asp and Glu, which define subfamilies SNRK2a and SNRK2b, respectively (Halford and Hardie, 1998
Interestingly, some members of the Arabidopsis (Arabidopsis thaliana) SNRK2 kinase family may function in controlling S limitation responses. Arabidopsis mutants defective for SNRK2.3 exhibit a slight decrease in the level of SULTR2;2 mRNA (encoding the low-affinity SO42– transporter) and elevated O-acetyl-Ser (precursor to Cys) accumulation (Kimura et al., 2006
The Arabidopsis kinase SNRK2.6 can be activated by two independent mechanisms, one that is ABA dependent and another that is ABA independent and stress dependent (Yoshida et al., 2006
Domain II of Arabidopsis SNRK2.6 is needed to elicit full stomatal closure and for the interaction with the PP2C-type phosphatase ABI1, and deletion of the rice (Oryza sativa) OSRK1 C terminus destroys the functionality of the protein (Chae et al., 2007
Some of the Chlamydomonas SNRK2 proteins (SNRK2.1, SNRK2.3, and SNRK2.4) have a specific feature that is not present in plant SNRK2 proteins: an inserted, nonconserved loop of 17 to 22 amino acids located immediately to the N-terminal side of the activation domain (Fig. 5). The activation domain contains the conserved Ser phosphorylation site (Johnson et al., 1996
SNRK2.1, SAC1, and SAC3 are the three genes that have been shown to have a regulatory role in the S deprivation responses of Chlamydomonas. While other mutants have been isolated that exhibit aberrant S deprivation responses, in most cases the gene responsible for the mutant phenotype has not been identified (Davies et al., 1994
Based on the phenotypes of the sac1 and ars11 mutants, SAC1 and SNRK2.1 have some similar regulatory features. However, SAC1 resembles animal Na+/SO4– transporters, such as SLT1 and SLT2 in Chlamydomonas (Davies and Grossman, 1998
Strains, Culture Conditions, and Mating
Chlamydomonas reinhardtii strains used in this work were D66 (nit2– cw15 mt+; Pollock et al., 2003
The sac3 mt+ mutant was crossed with the parental strain, 21gr (mt–), to obtain a sac3 mt– strain. The mating type of the ars constitutive progeny (sac3 phenotype) was determined by a PCR method (Werner and Mergenhagen, 1998
Cell wall-less strains D66 and ars11 were transformed by electroporation (Shimogawara et al., 1998
The selectable marker gene used for mutant generation was AphVIII under the control of the PSAD promoter and terminator; this gene confers paromomycin resistance to transformants (Sizova et al., 2001
ARS activity was visualized directly on agar plates (Davies et al., 1994
Genomic DNA and total RNA were isolated according to previously described methods (Schloss et al., 1984
RNA samples were treated with DNase (Qiagen; catalog no. 79254) and further cleaned using RNeasy columns (Qiagen) following the manufacturer's directions. Single-stranded cDNAs were synthesized from total RNA using an N-(polyT)20mer primer according to the SuperScript III RNaseH reverse transcriptase manual (Invitrogen). This cDNA population was used as a substrate for real-time PCR with gene-specific primer pairs; the single-stranded cDNAs were diluted 5-fold prior to inclusion in the reaction mixture. The CDS of SNRK2.1 was obtained by RT-PCR using the specific primers 5'-GCCTTCTTGCGACTGCCATACG-3' and 5'-CCTCAGTCGTTCATGCCGAA-3'. 3' RACE for SNRK2.1 transcripts was performed using the specific 5' primer 5'-GTTCCGTGAGGACCTACCCGAG-3' and a previously described polyT-Qt primer that allowed semi-nested PCR amplifications (Frohman, 1990
The ble gene from pSP124S (Lumbreras et al., 1998
Real-time PCR was performed using the Chromo4 thermocycler (Bio-Rad). Individual reactions had 25 µL final volume, consisting of 10 µL of DyNAMO SYBR Green quantitative PCR (qPCR) reagent (Finnzymes), 3.7 pmol of each primer, 1 to 2 µL of single-stranded cDNA (5-fold diluted from the reverse transcriptase reaction), and distilled water to 25 µL. The Chromo4 run protocol was as follows: denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 94°C for 10 s, annealing at 60°C for 30 s, and amplification at 72°C for 30 s, and fluorescence measurement after 80°C for 15 s. This last step avoids background signals that can result from the formation of primer dimers. The specificity of the PCR amplification was evaluated by a melting curve program (60°C–100°C, with a heating rate of 0.5°C s–1 and continuous fluorescence measurements) and electrophoretic analysis on 4% agarose gels. We used the CBLP gene as a housekeeping gene control (Chang et al., 2005
Sequences were analyzed using DNAstar software version 4.05 (Lasergene Navigator), BioEdit Sequence Alignment Editor version 5.0.9 (Department of Microbiology, North Carolina State University), the National Center for Biotechnology Information BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/), and Chlre3 (version 3) of the Chlamydomonas genome generated by the JGI (http://genome.jgi-psf.org/Chlre3/Chlre3.home.html). Sequence data for SNRK2.1 cDNA v1 has been deposited with the EMBL database under accession number AM900768.
The following materials are available in the online version of this article.
We thank Dr. Jeffrey Moseley for critical reading of the manuscript and for providing pJM43 vector and the JGI for generating a draft Chlamydomonas genome sequence, which has been invaluable for both gene identification and mutant analyses. We would also to dedicate this manuscript to Winslow Briggs in honor of his 80th birthday; Winslow has been both an enormously supportive colleague and an inspiration to all members of the Grossman laboratory. Received January 9, 2008; accepted March 2, 2008; published March 7, 2008.
1 This work was supported by National Science Foundation Grant MCB 0235878 awarded to A.R.G. and by the Carnegie Institution, the Ministerio de Educación y Ciencia (Spain), and the Marie Curie OIF-6 (European Union).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: David Gonzalez-Ballester (davidg3{at}stanford.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.116137 * Corresponding author; e-mail davidg3{at}stanford.edu.
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TOP
ABSTRACT
RESULTS
Ct method [2–(Ctsample – Ctcontrol gene)] using the CBLP amplification product as an internal standard. The primer pairs used for qPCR were as follows: 5'-CTTCTCGCCCATGACCAC-3' and 5'-CCCACCAGGTTGTTCTTCAG-3' for CBLP, 5'-CGCGCCGTCACTTGTTTGTTG-3' and 5'-GCCCACTTCTTTACCCAGCACCTC-3' for ARS1, 5'-ACGACGCCATGGACAACATGTAC-3' and 5'-ACCCAGTGCGCTCCGTTCAG-3' for SAC3, 5'-TCGGTACTTGCAGCTGAGGTTAGG-3' and 5'-ACACCGTCCAGCCCATGTATCTT-3' for the SNRK2.1 3' UTR, 5'-TATGGAGCGTGGGCGTTATCTTG-3' (underlined sequence binds to the beginning of exon VII, whereas the remaining 5' sequence of the primer binds to the end of exon VI) and 5'-CCTCCATGGTGATGCGCTT-3' for SNRK2.1 exons VI and VII, 5'-ACGTGGCATGCAGCTCAT-3' and 5'-CTTGCCACTTTGCCAGGT-3' for SULTR2, 5'-ACGGGTCTTCGAGCGAATTGC-3' and 5'-CGACTGCTTACGCAACAATCTTGG-3' for SLT1, 5'-CCTCGCTCTCCTCGCTGCTG-3' and 5'-CGGCCGACTTGGGTAATTGC-3' for ECP76, and 5'-GGACGGCAGCATCATGGTGAGC-3' and 5'-TCCACACGCCCTTGACCTTGAG-3' for SBDP. The sizes of the amplification products were between 100 and 300 bp.