Supplementary materials
Establishment and field applications of real-time PCR methods for the quantification of potential
MIB-producing cyanobacteria in aquatic systems
Zhongjie Wang1, Gaofei Song2, Jihai Shao3, Wenhua Tan4, Yeguang Li1, Renhui Li2*
1 Key Laboratory of Plant Germplasm Enhancement and Speciality Agriculture, Wuhan Botanical
Garden, Chinese Academy of Sciences, Wuhan, 430074, P. R. China
2 Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan,
430072, P. R. China
3 Resources and Environment College, Hunan Agricultural University, Changsha, 410128, P. R. China
4 Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans-Knöll-Straβe
8, D-07745 Jena, Germany
Email:
Jiezhongwang1@163.com (Zhongjie Wang)
sgf2016@126.com (Gaofei Song)
shaojihai@gmail.com (Jihai Shao)
tanwenhua_009@163.com (Wenhua Tan)
yeguang@wbgcas.cn (Yeguang Li)
reli@ihb.ac.cn (Renhui Li)
* Corresponding author:
Professor Renhui Li
E-mail: reli@ihb.ac.cn
Address: No. 7 Donghu South Road, Wuchang District, Wuhan, 430072, China
Tel: (+86) 27 68780067
Fax: (+86) 27 68780123
Fig. S1 Location of primers for qPCR assays.
Table S1 Characterization of the three investigated water bodies.
Qinghai
Water body
Donghu Lake
Lushui Reservoir
Lake
Location
Hubei
2
Hubei
2
Qinghai
Area
33 km
58 km
4583 km2
Salinity
freshwater
freshwater
1.25%
Average depth
2.5 m
10 m
21 m
Temperature
4.7-32.3 °C
6.6-32.2 °C
-0.9-22.3 °C
pH
7.5-9.0
7.7-8.9
8.4-9.1
T-N (mg/L)
0.27-10.5
0.74-1.37
8.5-13.1
T-P (mg/L)
0.006-0.790
0.01-0.18
< 0.01
Yes
Yes
No
VOCs
Mar-May
July-Oct
Local area
Sampling sites
11
6
20
Sampling time
Mar/2009-Dec/2010
July/2010-Nov/2011
May/2011
Sampling levels
0.5 m
0.5 m, 10 m, 15 m
0.5 m, 5 m
Source of drinking
water
Fig. S2 Location of sampling sites in three water bodies.
Fig. S3 Gel electrophoretic results of PCR reactions using qPCR primers. A: primers MIB-Rf/r; B:
primers CRTf/r. Lanes 1-7 are PCR products of Pseudanabaena sp. dqh15, Planktothricoides
raciborskii CHAB 3332, Planktothricoides sp. TH7, Leptolyngbya sp. A2 and three MIB-contained
environmental samples from Lushui Reservoir, Donghu Lake and Qinghai Lake respectively. Lanes
8-24 are cyanobacterial MIB-free strains Pseudanabaena sp. CHAB 759, Planktothricoides raciborskii
CHAB 3334, Planktohrix agardhii CHAB 166, Planktothrix pseudagardhii CHAB 366, Leptolyngbya
sp. Hp1, Leptolyngbya sp. Hp5, Lyngbya sp. CHAB 120, Oscillatoria sp. CHAB 1174, Oscillatoria sp.
CHAB 640, Phormidium sp. D6, Tychonema sp. CHAB 663, Arthrospira sp. CHAB 318,
Aphanizomenon issatschenkoi CHAB 473, Anabaena spirioides CHAB 508, Cylindrospermopsis
raciborskii CHAB 145, Nostoc sp. CHAB 1176 and
Calothrix sp. CHAB 2383.
Corresponding sequences:
Primers MIB-Rf/r
Pseudanabaena sp. dqh15: CGACAGCTTCTACACCTCCATGACACTAATCGACCCCATCGGAGGCTACGTCCTCCCAC
CAGATCTTTTCTTCGATCCGCGCGTCCGTCACACAGCCTTCCTAGCTGGGACGGCGGTCGTTCTGGTCAACGATCTC
CTTTCGGTTGCCAAAGATCTGGCAGACGAGCAGCCACCTGTCAACATGGTGCTACAGATTGCGGCG
Planktothricoides raciborskii CHAB 3332: CGACAGCTTCTACACCTCCATGACGCTAATCGACCCCATTGGAGGGTAC
GTCCTCCCAGGGGATCTTTTCTTCGAGCCGCGCGTCCGTCACGCAGCGTTCTTGGCCGGGATAGCCTCGCTCCTGGT
CAACGATCTCCTTTCCGTCAATAAGGATCTGGCAGACGAGCAGCCACCGGTCAACATGGTGCTACAGATTGCGGC
G
Planktothricoides sp. TH7: CGACAGCTTCTACACCTCCATGACGCTAATCGACCCCATCGGAGGGTACGTCCTCCCA
GGGGATCTTTTCTTCGAGCCGCGCGTCCGTCACGCAGCGTTCTTGGCCGGGATAGCCTCGCTCCTGGTCAACGATC
TCCTTTCCGTCAATAAGGATCTGGCAGACGAGCAGCCACCGGTCAACATGGTGCTACAGATTGCGGCG
Leptolyngbya sp. A2: CGACAGCTTCTACACCTCCATGACGCTGCTCGACCCCATCGGTGGGTACATCCTGCCGGCAG
ATCTCTTCTTCGAACCCCGTGTCCGTCACGCAGCGTTCTTGGCCGGGACGGCCGTCGTTCTGGTCAACGATCTTCTT
TCGGTCGCCAAGGATCTGGCGGACGAGAAGCCACCGGTCAACATGGTGCTACAGATTGCGGC
Primers CRTf/r
Pseudanabaena sp. dqh15: CTGTTACGCCACCTTCTTTATGTTCGTCTCTTGGAGTGCCTACGCAGCGTGGCGTTACA
CTGGCGAGTATCCGCCAGCGTGGAAGTATCTGGCTGCGCGACAGCACGACAGCTTCTACACCTCCATGACACTAAT
CGACCCCATCGGAGGCTACGTCCTCCCACCAGATCTTTTCTTCGATCCGCGCGTCCGTCACACAGCCTTCCTAGCTG
GGACGGCGGTCGTTCTGGTCAACGATCTCCTTTCGGT
Planktothricoides raciborskii CHAB 3332: CTGTTACGCCACCTTCTCTATGTTTGTCTCTTGGGGTGCCTATGCGGCAT
GGCGCTACACCGACGAGTACCCGCCAGCTTGGAAGTATCTAGCCGCGCGCCAGCACGACAGCTTCTACACCTCCA
TGACGCTAATCGACCCCATTGGAGGGTACGTCCTCCCAGGGGATCTTTTCTTCGAGCCGCGCGTCCGTCACGCAGC
GTTCTTGGCCGGGATAGCCTCGCTCCTGGTCAACGATCTCCTTTCCGT
Planktothricoides sp. TH7: CTGTTACGCCACCTTCTTTATGTTTGTCTCTTGGGGTGCCTATGCGGCATGGCGCTACA
CCGACGAGTACCCGCCAGCTTGGAAGTATCTAGCCGCGCGCCAGCACGACAGCTTCTACACCTCCATGACGCTAAT
CGACCCCATCGGAGGGTACGTCCTCCCAGGGGATCTTTTCTTCGAGCCGCGCGTCCGTCACGCAGCGTTCTTGGCC
GGGATAGCCTCGCTCCTGGTCAACGATCTCCTTTCCGT
Leptolyngbya sp. A2: CTGTTACGCCACCTTCTCTATGTTTGTCTCTTGGAGTGCCTATGCGGCGTGGCGCTACACCGA
CGAGTACCCTCCAGCCTGGAAGTACCTGGCTGCACGACAGCACGACAGCTTCTACACCTCCATGACGCTGCTCGAC
CCCATCGGTGGGTACATCCTGCCGGCAGATCTCTTCTTCGAACCCCGTGTCCGTCACGCAGCGTTCTTGGCCGGGA
CGGCCGTCGTTCTGGTCAACGATCTTCTTTCGGT
Fig. S4 Phylogenetic trees of the PCR products from environmental total DNA and cyanobacterial MIB
cyclase gene (mic) sequences from GenBank. Neighbor-Joining (NJ) and Maximum Likehood (ML)
algorithms were used. A: from MIB contained 2# site of Lushui Reservoir in July 2010 amplified by
primers CRT(f/r), B: from MIB contained QH25S site of Qinghai Lake in May 2011 amplified by
primers MIB-R(f/r). Homologous cyclase gene from Streptomyces coelicolor A3(2) was used as
outgroup.
Fig. S5 Spatiotemporal changes of mic copies in Lushui Reservoir during the period of July 2010 to
November 2011. A: quantified by TaqMan qPCR, B: quantified by SYBR Green qPCR. “*” represent
off-flavor event occurred and MIB was detected and quantified in this month.
Fig. S6 Spatiotemporal changes of total cyanobacteria in Lushui Reservoir during the period of July
2010 to November 2011.
Description: As shown in Fig. S5, mic genes were detected in most samples by the TaqMan qPCR, and
their abundance ranged from 103 to106 copies L-1. The site 2# in July 2010 contained the highest mic
densities as 1.94 × 106 copies L-1. On the whole, July-November in 2010 and 2011 are the two periods
with high densities of mic exceeding 105 copies L-1 in most sites. The abundance of mic is relatively low
(at the level of 104 copies L-1) during the period from December 2010 to June 2011, and the lowest
densities were detected in sites 2# (8.2 × 103 copies L-1) and 1S (9.18 × 103 copies L-1) in December 2010.
However, the variation of average total cyanobacteria is inconsistent with the abundance of mic copies.
The total cyanobacteria were shown to have higher abundances in the periods from July 2010 to April
2011 and September to November 2011, as cell abundance ranged from 4.08 × 107 to 2.07× 109 cells L-1.
Water samples analyzed for MIB contents by GC showed that all the sites in July-September 2010
contained MIB ranging from 16.3 to 45.3 ng L-1. Furthermore, MIB was also detected at some sites in
2011, such as 1# (14.8-29.8 ng L-1) and 6# (22.3 ng L-1) sites in February 2011; 1# (12.7-22.5 ng L-1) and
3# (7.2-10.2 ng L-1) sites in August-September 2011; 6# site in October 2011 (17.6 ng L-1). Combined
with the quantification by the qPCRs, it was revealed that MIB-detected samples were all with high
abundances of mic copies (over 105 copies L-1 level).
Fig. S7 Spatiotemporal changes of mic copies in Donghu Lake during the period of March 2009 to
December 2010. SYBR Green qPCR was used for the quantification of mic copies. “*” represent
off-flavor event occurred in this period.
Description: The spatiotemporal variations of mic gene copies in Donghu Lake, the largest urban lake in
China, were analyzed using SYBR Green qPCRs during the period from March 2009 to December 2010.
Two periods with higher mic abundances were shown at most sites, as one from March 2009 to April
2009 with the highest abundance (maximum 7.26 × 106 copies L-1) and the other from March 2010 to
May 2010 (maximum 1.32 × 106 copies L-1). Except the sites 5# and 6# in July 2009 and December 2010,
the mic densities were in the level of 103-104 copies L-1, and mic gene was not even detected in many sites,
such as most sites in May-June 2009. For the average total cyanobacteria, different variations were
presented. The period with higher cyanobacterial abundance corresponded to lower mic abundance and
vice versa in the monitoring period.
Fig. S8 Spatiotemporal changes of mic copies in Qinghai Lake in May 2011 using SYBR Green and
TaqMan qPCRs. “*” represent off-flavor event occurred and MIB was detected and quantified.
Fig. S9 Temporal changes of mic copies in 1# and 4# stratified sampling sites of Lushui Reservoir.
TaqMan qPCR was used for quantification.
Description: Quantification on mic abundances at stratified sites 1# and 4# along different layers (0.5 m,
10 m, 15 m) in Lushui Reservoir were performed. Except a few samples, mic abundances were
significantly different (P < 0.05) between different layers of the same site during the periods of
July-December 2010 and June-November 2011, and these periods were the seasons with high mic
abundances. It showed hardly differences among the layers at lower mic abundances during
February-May 2011.
Discussion: The distributional difference of cyanobacterial mic (potential MIB-producing cyanobacteria)
in vertical layers (0.5 m, 10 m and 15m) during the period of high mic abundances is an interesting
phenomenon. In shallow waters, movements of odor-producing organisms in different depths are
relatively frequent (Jüttner and Watson 2007). The highest geosmin concentrations were found to appear
at the surface (0.5 m) or bottom (10 m) in different months in Lake Schleinsee (Germany), and the
particle-bound geosmin could move to deeper layers in Lake Ontario due to the movement of
geosmin-producing organisms (Rao et al. 2003; Jüttner and Watson 2007; Watson et al. 2007). Present
study first reported that the distribution of MIB-producing cyanobacteria is highly relevant to its
abundance. The factors leading to this relationship is an interesting question worthy of further
investigation.
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