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Supplementary Information
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Materials and Methods
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Construction of mutant strains
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Sequences for all primers used for PCR amplification are listed in Table S3.
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Synechococcus elongatus PCC 7942
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(1) dnaA gene deletion mutants
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DNA regions up- and downstream of the dnaA gene were amplified by PCR using the
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primer
pairs
syfdnaA-us-F/syfdnaA-us-R
and
syfdnaA-ds-F/syfdnaA-ds-R,
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respectively; a kanamycin-resistance gene was amplified using primers syfdnaA-Km-F
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and syfdnaA-Km-R. Fragments were recombined by PCR using the primer pair
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syfdnaA-us-F and syfdnaA-ds-R and the product was used to transform Synechococcus.
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(2) Introduction of the FLAG-tagged dnaB gene into various mutants
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DnaB fused to three copies of the FLAG epitope tag at the C-terminus (Ohbayashi, et
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al., 2013) was expressed in wild-type and mutant strains.
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Synechocystis sp. PCC 6803
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(1) Thymidine kinase (TK) expression strain
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The TK gene was PCR-amplified using the primer pair TK-F-NdeI and TK-R-BglII with
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pNSHA::TK (Watanabe, et al., 2012) used as a template. The fragment was digested
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with NdeI and BglII and then inserted at the corresponding restriction sites of the
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pTCHT2031V (Ishizuka, et al., 2006) vector (referred to as pTCHTtk), which was used
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to transform Synechocystis, resulting in genomic insertion of the TK gene.
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(2) dnaA gene deletion mutant
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DNA regions up- and downstream of the dnaA gene were PCR-amplified using the
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primer
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respectively, and a kanamycin-resistance gene was amplified using primers
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syndnaA-Km-F and syndnaA-Km-R. Fragments were recombined by PCR using the
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primer pair syndnaA-us-F and syndnaA-ds-R and the product was used to transform
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Synechocystis.
pairs
syndnaA-us-F/syndnaA-us-R
1
and
syndnaA-ds-F/syndnaA-ds-R,
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(3) Predicted oriC region (POR) deletion mutant
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The full-length slr0964 gene containing POR was deleted as follows. DNA regions up-
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and
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slr0964-us-F/slr0964-us-R and slr0964-ds-F/slr0964-ds-R, respectively, and the
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spectinomycin-resistance gene was amplified using primers Spec-F and Spec-R. The
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three fragments were recombined by PCR using the primer pair slr0964-us-F and
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slr0964-ds-R and the product was used to transform Synechocystis.
downstream
of
slr0964
were
PCR-amplified
using
the
primer
pairs
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Anabaena sp. 7120
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(2) dnaA gene deletion mutant
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DNA regions up- and downstream of the dnaA gene were PCR-amplified using the
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primer pairs anadnaA-5F/anadnaA-5R and anadnaA-3F/anadnaA-3R, respectively. The
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fragments were cloned between the SacI/BamHI and BamHI/XhoI sites, respectively, of
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pHSG396 (Takara Bio Inc.). A neomycin-resistance cassette excised from plasmid
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pRL161 (Elhai & Wolk, 1988) by digestion with BamHI was inserted into the
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corresponding restriction site between the up- and downstream fragments. The
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SacI-XhoI fragment was excised from the resultant plasmid and inserted at the
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corresponding sites of pRL271 (Black, et al., 1993) to generate pRdnaAK, which was
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transferred by conjugation to Anabaena sp. PCC 7120 (Elhai, et al., 1997) to obtain the
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dnaA deletion mutant. Segregation was confirmed by PCR using the primer pair
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anadnaA-F and anadnaA-R.
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(2) TK expression strain
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The pATK plasmid was used to express TK in Anabaena PCC 7120. The TK gene was
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PCR-amplified using the primer pair TK-F and TK-R with pNSHA::TK (Watanabe, et
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al., 2012) used as a template. The fragment was digested with SalI and inserted between
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the SmaI and SalI sites of the shuttle vector pAM505 (Yoon & Golden, 1998) to
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generate pATK. Plasmid pATK-S, in which a spectinomycin-resistance cassette from
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plasmid pDW9 (Golden & Wiest, 1988) was inserted into the SalI site of pATK, was
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used to express TK in the dnaA mutant.
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Supplementary Reference
2
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Black TA, Cai Y & Wolk CP (1993) Spatial expression and autoregulation of hetR, a
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gene involved in the control of heterocyst development in Anabaena. Mol Microbiol 9:
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77-84.
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Elhai J & Wolk CP (1988) A versatile class of positive-selection vectors based on the
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nonviability of palindrome-containing plasmids that allows cloning into long
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polylinkers. Gene 68: 119-138.
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Elhai J, Vepritskiy A, Muro-Pastor AM, Flores E & Wolk CP (1997) Reduction of
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conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC
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7120. J Bacteriol 179: 1998-2005.
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Golden JW & Wiest DR (1988) Genome rearrangement and nitrogen fixation in
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Anabaena blocked by inactivation of xisA gene. Science 242: 1421-1423.
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Ishizuka T, Shimada T, Okajima K, Yoshihara S, Ochiai Y, Katayama M & Ikeuchi M
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(2006) Characterization of cyanobacteriochrome TePixJ from a thermophilic
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cyanobacterium Thermosynechococcus elongatus strain BP-1. Plant Cell Physiol 47:
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1251-1261.
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Yoon HS & Golden JW (1998) Heterocyst pattern formation controlled by a diffusible
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peptide. Science 282: 935-938.
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Supplementary Figure Legends
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Figure S1. DnaA expression level and protocol for generating dnaA disruptants.
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(a) DnaA protein level was determined in WT Synechococcus by western blotting. Cells
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were cultured until they reached stationary phase, then diluted to OD750 = 0.2; after
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cultivation for 18 h in the dark, the culture was transferred to the light condition (time 0)
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to restart cell growth. Cell extracts were separated by sodium dodecyl sulfate
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polyacrylamide gel electrophoresis, and the gel was stained with Coomassie Brilliant
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Blue (CBB) or analyzed by immunoblotting for DnaA (upper panel). (b) Deletion of the
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dnaA gene by homologous recombination and insertion of the kanamycin resistance
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(Kmr) gene into the dnaA locus. (c) Flow chart depicting the isolation of dnaA
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disruptants. Since cyanobacteria have a multicopy genome, PCR was used to ascertain
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complete segregation. The 1.6-kb DNA fragment containing the dnaA gene was
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amplified in the WT strain (white arrowhead), while this and a 1.2-kb DNA fragment
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(black arrowhead) were detected in the Kmr strain, indicating that gene deletion was
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incomplete in all clones (Colony PCR-1). Selected clones were transferred to medium
3
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containing Km and cultured until stationary phase to induce complete segregation of the
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WT dnaA gene. The 1.2-kb fragment alone was detected in 18 of 77 colonies in the
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second round of screening, indicating that in these clones all copies of the dnaA gene
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were replaced by the Kmr gene (Colony PCR-2).
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Figure S2. Genomic integration of pANL plasmid in dnaA disruptants.
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(a) Plasmid-inserted regions are indicated by a red bar in ΔdnaA-1 (left) and -2 (right)
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genomes; oriC is also shown. (b) Endogenous chromosome (Chr, upper) and plasmid
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(Plasmid, middle) sequences and results from capillary sequencing (lower) of a
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ΔdnaA-1 strain. Blue characters in the upper and middle parts and horizontal blue lines
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in the lower part indicate the sequence that is homologous between the chromosome and
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plasmid. The region downstream of GAAAATC and upstream of GATTTTC in the
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Synpcc7942_0826 gene that is homologous between the chromosome and pANL was
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changed to the plasmid sequence, as confirmed by capillary sequencing analysis. (c)
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Capillary sequencing data in a ΔdnaA-2 strain. P, plasmid; F, R forward and reverse
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sequencing primers, respectively, shown in Fig. 3A and B.
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Figure S3. Whole genome dnaB-binding activity.
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DnaB-binding activity in WT Synechococcus, ΔdnaA-1, and ΔdnaA-2 strains in which a
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FLAG-fused dnaB gene was inserted downstream of the native dnaB promoter.
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Synchronized cultures were transferred to light conditions and cultivated for 5 h, then
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subjected to chromatin immunoprecipitation (ChIP)–quantitative (q)PCR. (a) Genomic
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regions amplified by qPCR are represented as ORF ID. (b) WT and ΔdnaA-1 cell
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extracts were probed with antibodies against FLAG (upper) and RpoD1 (bottom;
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internal control) in the western blot analysis. (c) ChIP-qPCR analysis of cell extracts
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from each strain using an anti-FLAG antibody. Values are shown as percentage recovery
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of the total input DNA. Data represent the mean of three biological replicates; error bars
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indicate standard deviation.
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Figure S4. Cell viability by SYTOX Green (SG) staining.
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Cells from the 1-week time point in Figure 3a were stained with SG and analyzed by
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fluorescence microscopy. Brightfield with differential interference contrast (DIC),
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autofluorescence (AF), SG, and the merged AF and SG images (merge) are shown.
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SG-stained cells were counted as dead cells (white arrowheads).
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Figure S5. Up- or down-regulated genes in dnaA disruptant
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Genes up- or down-regulated in dnaA disruptants relative to WT. Total RNA was
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extracted from each strain after culturing for 4 weeks and analyzed by RNA-seq. Up- or
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down-regulated genes were calculated as RPKM value of dnaA disruptants vs WT; up >
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1.74, down < 0.57, respectively. Detailed gene lists are shown in Table S2.
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Figure S6. dnaA gene deletion and predicted oriC in Synechocystis sp. PCC 6803
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and Anabaena sp. PCC 7120.
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(a) Deletion of the dnaA gene. The Kmr gene was inserted into the dnaA locus in
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Synechocystis. (b) The insertion of the Kmr gene was confirmed by PCR using the
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primers indicated by arrows in (a). WTTK, wild-type strain; ΔdnaATK, dnaA disruptant.
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(c) Deletion of the slr0964 gene and POR containing a DnaA box. The
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spectinomycin-resistance gene (Specr) was inserted into the slr0964 locus. (d) The
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insertion of the Specr gene was confirmed by PCR using the primers indicated by
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arrows (1–4) in (c). Since bands of similar sizes were amplified using the primer set of 1
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and 2, insertion of Specr was confirmed with different primer sets (1, 3 or 1, 4). (e)
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Analysis of TK expression in the WTTK, ΔdnaATK, and ΔPORTK strains. Cell extracts
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were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the
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gel was stained with Coomassie Brilliant Blue (CBB) or probed with an antibody
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against HA (upper panel). (f) Number of cells under the same growth conditions as in
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Figure 4b. (g) The dnaA gene was deleted by inserting a neomycin resistance gene
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(Nmr) into the dnaA locus. (h) Insertion of the Nmr gene was confirmed by PCR using
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the primer set indicated by arrows in (g). WTTK, wild-type strain; ΔdnaATK, dnaA
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disruptant.
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Table S1. BLAST data base search for DnaA homologs.
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Table S2. Lists of up- or down-regulated genes in dnaA disruptants.
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Table S3. Primer list used in this study.
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