Accepted Manuscript A comparison of different methods for preserving plant molecular materials and the effect of degraded DNA on ddRAD sequencing Ying Guo, Guo-Qian Yang, Yun-Mei Chen, De-Zhu Li, Zhen-Hua Guo PII:
S2468-2659(18)30047-7
DOI:
10.1016/j.pld.2018.04.001
Reference:
PLD 104
To appear in:
Plant Diversity
Received Date: 9 March 2018 Revised Date:
17 April 2018
Accepted Date: 17 April 2018
Please cite this article as: Guo, Y., Yang, G.-Q., Chen, Y.-M., Li, D.-Z., Guo, Z.-H., A comparison of different methods for preserving plant molecular materials and the effect of degraded DNA on ddRAD sequencing, Plant Diversity (2018), doi: 10.1016/j.pld.2018.04.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Research Article
2
A comparison of different methods for preserving plant
3
molecular materials and impacts of degraded DNA on ddRAD
4
sequencing
RI PT
1
5
Ying Guo a,b , Guo-Qian Yang a,b , Yun-Mei Chen a,b , De-Zhu Li a* , and Zhen-Hua
7
Guo a*
SC
6
a
Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of
10
Sciences, Kunming 650201, China
11
b
12
*Author for correspondence.
13
Kunming Institute of Botany, Chinese Academy of Sciences
14
132 Lanhei Road, Kunming, Yunnan 650201, China
15
E-mail:
[email protected] ,
[email protected]
16
Tel: +86-871-65223503
17
Fax: +86-871-65223153
18
AC C
9
M AN U
8
EP
TE D
University of Chinese Academy of Sciences, Beijing 100049, China
ACCEPTED MANUSCRIPT Abstract Getting well-conditioned plant materials for multitudinous experiments is
20
difficult in many research projects, so exploring how to preserve materials is of special
21
importance after they were collected in case that biological macromolecules like DNA to
22
be degraded. Although some researches have demonstrated that DNA degradation has little
23
effect on some traditional molecular markers, the impacts of DNA degradation on ddRAD-
24
seq, a popular reduced-representation sequencing technology, have not been adequately
25
investigated. In this study, we first chose six woody bamboo species (Bambusoideae,
26
Poaceae) to explore appropriate methods for preserving molecular materials with two DNA
27
extraction approaches. Then we sequenced another twenty-one bamboos and examined the
28
impacts of DNA quality on data generation using ddRAD-seq technique (MiddRAD-seq).
29
Finally, we reconstructed phylogenies of twenty woody bamboo species. We found dry-
30
powdered DNA had the longest preserving time compared with TE-dissolved DNA which
31
were extracted with both modified CTAB protocol and DNAsecure plant kit. ddRAD-seq
32
was robust except that DNA was severely degraded and we also demonstrated the
33
systematic positions of the sampled Phyllostachys species. Our results suggest that dry-
34
powdered DNA is a commendable way to preserve molecular materials. Furthermore, DNA
35
degradation to moderate level has little effect on reduced representation sequencing
36
techniques represented by ddRAD-seq.
SC
M AN U
TE D
EP
AC C
37
RI PT
19
38
Keywords molecular materials; DNA extraction; DNA preservation; DNA quality;
39
ddRAD-seq; bamboo; phylogeny
40 41
ACCEPTED MANUSCRIPT 42
1.
Introduction Collecting experimental materials is the necessary prerequisite for almost every
44
biological research project. It’s common that researchers collect plant materials from
45
different locations or even from different countries in many cases. As some plants are
46
endemic species which are difficult to get samples, it’s of crucial importance to preserve
47
their biological samples in an appropriate way once obtained (Doyle and Dickson, 1987).
48
DNA extracted from plant materials is often degraded to varying degrees and it may be
49
adverse to future researches. However, few researches lay stress on this problem and
50
most of these researches were designed for one species or different varieties with a short
51
preserving time ranging from few hours to few months, so the conclusions drawn were
52
not very convictive, especially for some plants that degraded slightly in a short time
53
(Liang et al., 2016).
TE D
M AN U
SC
RI PT
43
Degraded genomic DNA may have little effect on some traditional molecular
55
markers, such as microsatellites (Ledoux et al., 2013; Prugh et al., 2005; Qin et al., 2017;
56
Scandura et al., 2006). However, with the application of next-generation DNA
57
sequencing (NGS), more and more reduced representation sequencing methods were used
58
in recent molecular researches, such as molecular phylogenetics and molecular ecology
59
(Baird et al., 2008; Peterson et al., 2012; Poland et al., 2012; Yang et al., 2016).
60
Restriction-site associated DNA sequencing (RAD-seq) was first introduced in 2008 as a
61
rapid SNP discovery and genotyping method (Baird et al., 2008). This technology utilizes
62
RAD-tags which are short DNA fragments adjacent to a particular restriction enzyme
63
recognition site to reflect the sequence characteristics of the whole genome and construct
AC C
EP
54
ACCEPTED MANUSCRIPT sequencing library for high-throughput sequencing (Baird et al., 2008; Peterson et al.,
65
2012). Although it is originally designed for intraspecific genomic analysis, many recent
66
studies which involve in many fields, such as phylogenetic analysis, population genomics,
67
ecological and evolutionary genomics, have proved that it can also be useful at
68
interspecific levels (Andrews et al., 2016; Cariou et al., 2013; Cruaud et al., 2014; Rubin
69
et al., 2012; Takahashi et al., 2014; Wang et al., 2013). Nevertheless, as a relatively new
70
technology, there are few researches about the impacts of DNA degradation on this
71
technology. Tin et al (2014) and Graham et al (2015) used few animal specimens or fresh
72
tissues (ants and whitefish) to investigate this problem respectively. However, as the
73
research objects of these two studies are animals, it is necessary to fill this gap in plants
74
because of the essential difference between animals and plants materials.
M AN U
SC
RI PT
64
The Bambusoideae (bamboos), including more than 1400 described species in nearly
76
120 genera, is one of the most important member of Poaceae (Akinlabi et al., 2017; BPG,
77
2012), and has great ecological and economic value as it provides food and raw materials
78
for construction and manufacturing, especially woody bamboos (Li et al., 2006).
79
Bambusoideae is also a difficult group in taxonomy as their complex polyploidy
80
evolutionary history and slow evolutionary rate (Triplett et al., 2014; Zhang et al., 2012).
81
Although Bambusoideae has been extensively studied in evolutionary genetics contexts, its
82
phylogenetic relationship is still difficult to resolve (Wysocki et al., 2016; Zhang et al.,
83
2016). In recent years, analyses based on these RAD tags provide an opportunity to yield
84
robust phylogenetic inferences on bamboo phylogenetics (Wang et al., 2017; Wang et al.,
85
2013). The woody bamboos are widely distributed around the world, from Asia, America
AC C
EP
TE D
75
ACCEPTED MANUSCRIPT to Africa (Akinlabi et al., 2017; Bamboo, 2012). Therefore, it is a good model for
87
conducting researches on plant material preservation. Here, we applied two DNA
88
extraction methods and four different preservation methods on 3 temperate woody
89
bamboos and 3 tropical woody bamboos to explore the appropriate preservation method for
90
plant materials monitored as longest up to three years. Besides, we used the modified
91
ddRAD-seq (MiddRAD) with 21 woody bamboos to examine the effect of DNA quality on
92
RAD-seq. Our main goals were: (1) to find an appropriate preservation method for
93
precious plant materials in order to obtain high quality DNA for subsequent DNA analysis,
94
and (2) to examine the impact of degraded DNA on ddRAD-seq approach (MiddRAD-seq)
95
using the STACKS bioinformatics pipeline (Catchen et al., 2013; Yang et al., 2016).
96 2.
Materials and Methods
98
2.1 Plant Materials and Treatments
TE D
97
M AN U
SC
RI PT
86
Six bamboo species including three temperate woody bamboos (Phyllostachys edulis,
100
Indosasa hispida cv. rainbow, Acidosasa purpurea) and three tropical woody bamboos
101
(Dendrocalamus latiflorus, Bambusa multiplex cv. Alphonse-Karr, B. emeiensis) were
102
chosen to explore the ideal plant leaf material preservation method with observing time
103
as long as three years. Fresh leaves from each bamboo species were collected and divided
104
into three equal parts (Replicate1, Replicate2, and Replicate3). Replicate1 was dried with
105
silica gel at room temperature (RT), while Replicate2 was sealed within zip-lock bags
106
and stored in -80
107
two different methods (the modified CTAB protocol and DNAsecure plant kit)
AC C
EP
99
refrigerator. Replicate3 was used for DNA extraction directly with
ACCEPTED MANUSCRIPT 108
respectively. Then total genomic DNA extracted from Replicate3 was divided equally
109
into ten tubes, of which five of them were dissolved in TE solution and another five tubes
110
were dried by a freeze drier to get dry-powdered DNA. Finally, all ten tubes were stored
111
in -80
112
material which stored in low temperature (LT, Replicate1) and RT (Replicate2) were
113
extracted and detected with electrophoresis and Nanodrop respectively. After a year of
114
observation, we found that there was little difference between detection results of six-
115
months storage and twelve-months storage. So we extended the detection period to every
116
12 months. Considering the possible longer preserving time of dry-powdered DNA, we
117
extended the detection period of it to 24 months. If the DNA was detected degraded, this
118
sample was re-extracted and detected again to confirm the degradation. The whole
119
experimental flowchart is shown in Fig. 1. Fresh leaf materials of these six species were
120
all collected from plants grown in Kunming Institute of Botany, Chinese Academy of
121
Sciences (KIBCAS) (N25°07′04.9″, E102°44′15.2″).
TE D
M AN U
SC
RI PT
refrigerator. Every six months later, DNA from Replicate3 were detected and the
Furthermore, we sequenced 21 temperate woody bamboos (I. singulispicula has two
123
individuals) which were collected from different locations with ddRAD-seq (Yang et al.,
124
2016) to examine the impact of DNA quality on RAD-seq (Table 1). DNA of some
125
species were extracted immediately with fresh samples, some were extracted after
126
different preserving time, detailed information was shown in Table 1.
127 128 129
AC C
EP
122
2.2 DNA extraction and detection Total genomic DNA was extracted from leaf material using two different DNA extraction methods. One is DNAsecure plant kit (Tiangen Biotech, Beijing, China, DP320)
ACCEPTED MANUSCRIPT following the manufacturer’s protocol. The other one is a modified CTAB procedure
131
(Doyle, 1987). Specific steps are as follows. Firstly, the mortars were washed and dried
132
before starting the experiment; then the mortars were burned to sterilize using alcohol;
133
after which, 20 mg samples with moderate quartz sand were put in liquid nitrogen and
134
grounded quickly; then the grounded powder were transferred into a 2ml clean
135
microcentrifuge tube and were mixed immediately with 1ml of 4×CTAB extracting
136
solution to which 1% of β-mercaptoethanol (BME) had been added; then the samples
137
were placed in the 65
138
added, mixed and centrifuged at 9000 rpm when the tube was cooled to room temperature,
139
then the supernatant was transferred in another 2ml clean microcentrifuge tube and this
140
step was repeated once again; then the supernatant was mixed with 0.7 volumes of
141
isopropanol and the solution was incubated in -20
142
centrifuged at 10000 rpm for 8 minutes, the supernatant liquor was discarded and the
143
DNA was cleaned with 70% and absolute ethyl alcohol twice respectively, then dry
144
powdered DNA was produced by putting the tube in vacuum centrifuge concentrator at
145
50
146
and incubated the mixture with 0.5ul RNase at 37
147
solution was stored in -80 .
SC
M AN U
water for 1h; 1ml of chloroform-isoamyl alcohol (24:1) was
EP
TE D
for 1h; after that, the solution were
for 3-5 minutes; after that, we dissolved the dry-powdered DNA in 50ul TE solution
AC C
148
RI PT
130
for 1.5-2h to digest RNA; finally, the
The integrality of total genomic DNA was detected by agarose gel electrophoresis
149
while
150
spectrophotometer (Thermo Fisher Scientific, Delaware, USA).
151
concentration
and
purity
of
DNA
was
2.3 Construction and sequencing of the ddRAD libraries
detected
by
NanoDrop1000
ACCEPTED MANUSCRIPT We used Qubit 2.0 (Thermo Fisher Scientific, Delaware, USA) to detect the
153
concentration of total genomic DNA and diluted DNA to the proper concentration (40
154
ng/ul). Because of the different preserving time and methods, the samples were
155
sequenced in different sequencing batches. ddRAD libraries were prepared according to
156
Yang et al (2016). Each sample was digested with two enzymes, i.e. Ava
157
600-700 base pair (bp) DNA fragments were selected from agarose gel and recovered by
158
E.Z.N.A DNA gel extraction kit (D2500-02). We sequenced all ddRAD libraries on the
159
Illumina HiSeq X10 (Illumina, San Diego, CA, USA) by employing paired-end 150bp
160
sequencing mode at the Cloud Health Genomics Company (Shanghai, China).
SC
and MspI.
M AN U
161
RI PT
152
2.4 Data analysis
Clean data were obtained after two processing steps. Firstly, raw data were de-
163
multiplexed by process_radtags program implemented in STACKS version 1.41 (Catchen
164
et al., 2013; Catchen et al., 2011) and the sequence quality of each sample was checked
165
using FastQC version 0.11.2 (Andrews, 2014). Then, adapter reads and low-quality bases
166
which were below a Phred score of Q10 were deleted and the sequences were truncated to
167
a final length of 140bp with the process_radtags program. After reads trimming, ustacks
168
program was used to merge short-read sequences into tags/loci with ranging settings for
169
minimum depth of coverage (m = 5~15) and a maximum of 5-bp difference allowed
170
between stacks (M = 5). Then cstacks program was used to merge loci into catalog with
171
fourteen mismatches allowed between sample loci (n = 14). The sstacks program was
172
applied to match loci from an individual against the catalog built by cstacks and loci that
173
matched more than one catalog locus were excluded. Finally, the populations program
AC C
EP
TE D
162
ACCEPTED MANUSCRIPT was used to output single nucleotide polymorphism markers (SNPs) in phylip format.
175
After that, we used custom shell commands to compute the RAD tags number and
176
unexpected enzyme cutting site ratio for each sample. To determine and compare the
177
mapping ratio of reads to the genome, clean data of each individual was mapped to Ph.
178
edulis genome scaffolds (Zhao et al., 2014) with Bowtie 2.2.9 respectively (Langmead
179
and Salzberg, 2012).
RI PT
174
Finally, the data set was analyzed with a maximum-likelihood method using the
181
general time-reversible (GTR) model, which was implemented in RAxML-HPC BlackBox
182
version 8.2.10 on the CIPRES Science Gateway web server, with a rapid bootstrapping
183
analysis of 1000 bootstrap replicates (Stamatakis, 2014).
M AN U
SC
180
184 3.
Results
186
3.1 Exploring appropriate methods for preserving DNA materials
187
3.1.1 Initial DNA quality detection
TE D
185
Initial total genomic DNA was extracted from fresh leaves of six woody bamboos
189
using two methods and detected by agarose gel electrophoresis and spectrophotometer
190
respectively. The electrophoresis result showed that total genomic DNA extracted by
191
modified CTAB method all had clear main bands (Fig. 2A-F), while slight degradations
192
were found for DNA extracted by DNAsecure plant kit (Fig. 2G-L). The absorption ratio
193
of DNA at 260nm and 280nm were all between 1.8 and 2.0 while the DNA concentration
194
of samples extracted by modified CTAB method was higher than DNA extracted by
195
DNAsecure plant kit (Table 2).
AC C
EP
188
ACCEPTED MANUSCRIPT 196
3.1.2 The impact of different preserving methods and time on DNA quality Firstly, total genomic DNA was extracted with the modified CTAB protocol from leaf
198
materials stored under room temperature (RT, in silica gel) and low temperature (LT, fresh
199
leaves) for 6months, 12 months, 24 months and 36 months respectively. The
200
electrophoresis results showed that DNA in 6 months all had clear main band with no
201
obvious degradation or slight degradation (Fig. 3a), DNA degradation increased in 12
202
months for RT-stored materials and LT-stored materials (Fig. 3b), while some individuals
203
showed unclear main DNA bands and obvious degradations after 24 months storing (Fig.
204
3c, A L -F L ). Two individuals had degraded completely after 36 months storage, with one
205
(Fig. 3d, A R ) extracted from RT leaf material and the other one (Fig. 3d, B L ) extracted
206
from LT leaf material.
M AN U
SC
RI PT
197
Secondly, total genomic DNA was extracted with DNAsecure plant kit from leaf
208
materials stored under RT (in silica gel) and LT (fresh leaves) after storing 6 months, 12
209
months, 24 months and 36 months respectively. The electrophoresis results showed that
210
DNA in 6 months all had clear main band with slight degradation for LT-stored materials
211
(Fig. 4a). DNA degradation increased in 12 months for RT-stored materials and LT-stored
212
materials (Fig. 4b). Moderate DNA degradation for most RT-stored materials and LT-
213
stored materials were found after 24 months of storage (Fig. 4c), of which three
214
individuals had ambiguous main DNA bands (Fig. 4c, G R , I R , J R ). One individual has
215
degraded completely (Fig. 4d, G R ) and many individuals have unclear bands (degraded
216
moderately) after 36 months. On the other hand, the brightness of DNA bands in Fig. 4
217
was lower than that in Fig. 3 under the same condition, which might indicate the CTAB
AC C
EP
TE D
207
ACCEPTED MANUSCRIPT 218
method is more efficient than the DNAsecure plant kit method in improving DNA
219
concentration. Thirdly, total genomic DNA extracted from fresh bamboo leaves which were stored in
221
TE solution was detected after being preserved for 6 months, 12 months, 24 months and 36
222
months respectively. The electrophoresis results showed that DNA main bands were clear,
223
bright and only slight degradations were found after storing 6 months and 12 months
224
respectively (Fig. 5a, b). degradation increased in 24 months for DNA extracted by
225
modified CTAB method and the DNAsecure plant kit (Fig. 5c). Furthermore, four
226
individuals had ambiguous main bands and obvious degradations after 36 months storing
227
and total genomic DNA of these four individuals were all extracted by modified CTAB
228
method (Fig. 5d, C-F).
SC
M AN U
At last, dry-powdered DNA which stored in -80
was detected after 6 months, 12
TE D
229
RI PT
220
months and 36 months respectively. The electrophoresis result showed that main DNA
231
bands were clear, bright and only slight degradations were found after 6-36 months of
232
storage (Fig. 6). As the image of 36 months was taken with a different UV-
233
spectrophotometer, this might bring difference to the brightness of images even under the
234
same exposure rate. Even though this image was darker than other two images (Fig. 6a, b),
235
we could learn the high integrity of 36-months-preserved DNA by comparing it to the
236
DNA Marker. Nanodrop spectrophotometer detection also verified the high concentration
237
and purity of total genomic DNA at different preserving time (Appendix: Table A).
238
3.2 DNA quality evaluation through ddRAD sequence analysis
AC C
EP
230
ACCEPTED MANUSCRIPT To determine the quality of DNA preserved under different preserving time, we
240
sequenced and analyzed two I. singulispicula individuals with MiddRAD-seq (Table 1).
241
One (I. singulispicula 12162) was collected from Xishuangbanna, Yunnan province in
242
2012 (N21°48′51.18″E101°22′51.12″, elevation 568 m), while the other (I. singulispicula
243
16001) was collected from the same place in 2016. The electrophoresis result showed that
244
DNA extracted from leaf of I. singulispicula 12162 was completely degraded while the
245
other individual had clear band with only slight degradation (Fig. 7, No.1-2). We
246
estimated the data quality of these two individuals using FastQC software and shell
247
commands. The results showed the average reads quality of I. singulispicula 12162 had
248
no obvious difference with I. singulispicula 16001, but the raw reads number, clean reads
249
number and tags number of I. singulispicula 16001 were ten times larger than I.
250
singulispicula 12162 (Fig. 8a, d, e, f). Moreover, there were more reads which had
251
unexpected restriction enzyme cutting site and less reads which could be mapped to
252
Phyllostachys edulis reference genome in I. singulispicula 12162 than the other
253
individual (I. singulispicula 16001) (Fig. 8b, c). In summary, the data quality of I.
254
singulispicula 12162 was clearly worse than I. singulispicula 16001 and it was supposed
255
to be unfit for subsequent phylogenetic analyses.
SC
M AN U
TE D
EP
AC C
256
RI PT
239
Besides, we evaluated data quality of twenty more bamboo species from different
257
batches of ddRAD sequencing runs (Table 3). Among them, two species were extracted
258
with fresh materials immediately and sequenced within one month after sample collection
259
(Table 1, No. 2-3), twelve species were sequenced after their DNA dissolved in TE and
260
stored in -80
for 36 months (Table 1, No. 4-15) and another six species were sequenced
ACCEPTED MANUSCRIPT 261
after their leaves stored in silica gel for 36 months (Table 1, No. 16-21). The
262
electrophoresis result showed that DNA extracted from fresh leaves (Fig. 7, No. 2-3)
263
were nearly intact, while DNA dissolved in TE and stored in -80
264
slight degradation and DNA extracted from silica gel stored leaf materials (Fig. 7, No.
265
16-21) had moderate degradation. Meanwhile, all of them had clear main DNA bands. We
266
found the data of these twenty species were all of high quality. The data size of them was
267
ranged from 1.28 G to 6.36 G (Table 3). Tags number was all between 150000 and
268
210000, unexpected enzyme cutting site ratio of most species (except I. singulispicula)
269
were below 10% (Table 3). As expected, alignment ratios of species in genus
270
Phyllostachys to the reference genome were higher than that of species in other genus
271
and data missing ratio were usually lower than that of other species (except Ph. nigra)
272
(Table 3).
RI PT
SC
M AN U
TE D
273
(Fig. 7, No. 4-15) had
3.3 SNP discovery and phylogenetic reconstruction of temperate woody bamboos We then tried to use clean reads of these twenty species to reconstruct the phylogeny
275
of temperate woody bamboos with the Stacks software. Eleven data sets, ranging from
276
11904 SNPs (p=15) to 914416 SNPs (p=5), were yielded and used for phylogenetic
277
analyses with the maximum likelihood method. Topologies of phylogenetic trees which
278
constructed from different data sets were largely congruent except low MLBS (Maximum
279
Likelihood Bootstrap) of some nodes (Table 4). Phylogenetic analysis using 21063 SNPs
280
data set (p=14) revealed robust support for the relationships between twenty species
281
(100% MLBS, Fig. 9). Two clades were found. The first contained five species, of which
282
four of them were native to Japan (Suzuki, 1978) and clustered together, sister to I.
AC C
EP
274
ACCEPTED MANUSCRIPT 283
singulispicula. Within the first clade, Sasa bitchuensis was sister to S. ramose, forming a
284
clade
285
Semiarundinaria fortis. All the Phyllostachys members we used in this study were
286
contained in the second clade with high support (100% MLBS), which agreed well with
287
the phylogeny reported by Wang et al (2017). Two subclades were recognized in this
288
clade: the first one contained six species which all belonged to Phyllostachys sect.
289
Phyllostachys, while the second subclade (Phyllostachys sect. Heterocladae) contained
290
another nine species, which largely agreed with the morphology-based taxonomy (Fig. 9).
291
Notably,
292
aureosulcata, Ph. bissetii) which had ambiguous systematic positions before were all
293
clearly resolved in this study with 100% MLBS.
is
sister
to
another
clade
which
contains
Pleioblastus
chino
and
five
species
(Ph.
Discussion
nigra,
Ph.
robustiramea,
Ph.
varioauriculata,
Ph.
TE D
294
M AN U
SC
RI PT
that
295
4.
296
4.1 Appropriate methods for extracting and preserving leaf materials It is known that the purine and pyrimidine of nucleic acid have conjugated double bond,
298
which has a strong absorption effect on the ultraviolet ray. 230nm, 260nm and 280nm is
299
maximum absorption peak of carbohydrate, nucleic acid, protein and phenols respectively.
300
The ratio between them can be used to evaluate the purity of nucleic acid samples. The
301
absorption ratio of 260nm to 280nm (A260/280) indicates high quality of DNA when it is
302
between 1.8 and 2.0. When the ratio is higher than 2.0, it indicates that there is RNA
303
pollution in the extracted DNA, while if it is lower than 1.8, the samples might contain
304
small molecular pollutions such as protein or phenolic substances (Liang et al., 2016).
AC C
EP
297
ACCEPTED MANUSCRIPT 305
From the electrophoresis and spectrophotometer results, we could learn the initial total
306
genomic DNA extracted from fresh leaves by two DNA extraction methods was of high
307
quality and could be used for subsequent analysis (Table 2, Fig. 2). We found that DNA began to degrade obviously after 12 months for RT material and
309
12 months for LT material. The difference between DNA extracted from RT material and
310
LT material after 36 months storing was indistinctive (Fig. 3, Fig. 4), which indicate that
311
preserving leaf materials in silica gel or cryopreservation is not an efficient method to
312
protect DNA from being degraded after a long-term storage time. This result is out of our
313
expectation and we suppose it is probably due to complex secondary metabolites in plant
314
leaves. The difference between these two preserving methods would be evident after a
315
longer storage time. A comparison of the preserved effect of dry-powdered DNA and TE-
316
dissolved DNA (Fig. 5, Fig. 6) revealed that the former had only slight degradation after
317
36 months storing while the latter had obvious degradation after the same storage time,
318
which means dry-powdered DNA could be preserved longer than TE-dissolved DNA, and
319
dry-powdered DNA could be preserved at least 36 months without severe degradation. A
320
comparison of the two extracting methods indicates that the purity of DNA extracted by
321
two methods was both good, but the concentration of DNA extracted by the modified
322
CTAB procedure is clearly higher than the other method (Table 2, Table A). However, we
323
found that the DNA dissolved in TE solution of four individuals which had obvious
324
degradation after 36 months storage were all extracted by the modified CTAB method (Fig.
325
5). We detected them again to exclude the possible error of operation. This phenomenon
AC C
EP
TE D
M AN U
SC
RI PT
308
ACCEPTED MANUSCRIPT might indicate that DNA extracted by CTAB method had shorter preserving time than the
327
DNAsecure plant kit method, but it still need to be confirmed with longer observation time.
328
Many previous researches were performed on animals or bacteria using one or few
329
species with a short observing time (Dillon et al., 1996; Gray et al., 2013; Maxine and
330
Andrea, 2003; Mitchell and Takacsvesbach, 2008). Compared to them, our study adopts
331
more plant species and a more detailed experimental design and longer observation time,
332
so our results could provide useful suggestions for plant materials preservation. We could
333
conclude that: (1) The CTAB procedure is an appropriate method for extracting DNA from
334
fresh leaves of bamboos and even all grass plants as more high-quality DNA were obtained
335
than the DNAsecure plant kit procedure and the low reagent cost under the same
336
experimental condition; (2) The quality of DNA extracted from leaf materials stored in RT
337
had no obvious difference with the DNA extracted from materials stored in LT after 12
338
months (both moderately degraded); (3) Dry-powdered DNA is preserved better than TE-
339
dissolved DNA, which may severely degraded after 36 months.
340
4.2 ddRAD sequencing for degraded DNA sample
EP
TE D
M AN U
SC
RI PT
326
Until now, only few researchers have studied the impacts of degraded DNA for RAD-
342
seq. Tin et al used nine animal specimens (three ant species and six Hawaiian Drosophila
343
species) with significantly degraded DNA for RAD-seq and found degraded DNA might be
344
workable for RAD-seq, nevertheless, this study did not mention to which degree of
345
degraded DNA could affect RAD-seq (Tin et al., 2014). While another similar research
346
utilized a modified ddRAD-seq method (3RAD) on 8 individual lake whitefish (Coregonus
347
clupeaformis) following different treatments and demonstrated that highly degraded DNA
AC C
341
ACCEPTED MANUSCRIPT would affect the results of 3RAD-seq while moderate degraded DNA had little effect on
349
this approach (Graham et al., 2015). When compared to them, our study investigated the
350
influence of degraded DNA on MiddRAD-seq with 21 bamboo species and found that there
351
was no remarkable difference on data quality of the twenty species which had clear main
352
DNA bands, while the data quality of one individual (I. singulispicula 12162) with no
353
main DNA band was clearly worse than others. This phenomenon suggests that the
354
MiddRAD-seq approach is robust until total genomic DNA is severely degraded and DNA
355
degradation to moderate levels might be not a crippling factor for RAD-seq which is
356
similar to the conclusion of Graham et al (2015). As ddRAD arguably requires the highest
357
quality genomic DNA of all the RAD methods (Puritz et al., 2014), our conclusions could
358
also be applied to other reduced representation sequencing methods. However, as it is
359
possible to use moderately degraded DNA for RAD sequencing, we need to pay attention
360
to the problem that degraded DNA will not produce adequate data (Yang et al., 2016). And
361
duplicate removal is needed as libraries prepared from poor-quality DNA produced
362
thousands of possibly incorrect genotype calls (Tin et al., 2015). Our study here is the first
363
research which used plant materials to investigate the impacts of degraded DNA on
364
ddRAD-seq, and we believe that it will be very instructive for the wide application of
365
ddRAD-seq and other reduced representation sequencing methods in the plant kingdom.
366
4.3 SNP discovery and phylogenetic analysis
AC C
EP
TE D
M AN U
SC
RI PT
348
367
In this study, we reconstructed the phylogeny of twenty temperate woody bamboos
368
using the maximum likelihood method and their relationships were fully resolved with
369
high support (Fig. 9). Two clades were found, the first clade contained five species and the
ACCEPTED MANUSCRIPT systematic relationships of them coincided with morphological characters and the results
371
of previous molecular phylogenetic reports (Wang et al., 2017; Zhang et al., 2012). The
372
second clade contained all the Phyllostachys members and the relationships among them
373
were clearly resolved (100% MLBS). Remarkably, this is the first time we used ddRAD-
374
seq data to investigate the phylogeny of genus Phyllostachys and found it was maybe a
375
monophyletic group. However, only fifteen species (a total of 51 species in Flora of China,
376
2006) were used in our study, extending this approach to a broader taxonomic sampling is
377
needed to confirm the monophyly of genus Phyllostachys. Within this genus, two
378
subclades were produced. The first subclade contained six species of Phyllostachys sect.
379
Phyllostachys while the second subclade contained nine species. Four of them were placed
380
in Phyllostachys sect. Heterocladae with no doubt, other five species had uncertain
381
systematic positions, such as Ph. nigra. Ph. nigra was placed in sect. Phyllostachys in the
382
traditional classification, while Friar et al (1991) and Hodkinson et al (2000) suggested
383
that it should be classified into sect. Heterocladae based on their molecular phylogenetic
384
results with low support, and Hodkinson et al (2000) also pointed that this phenomenon
385
might be caused by hybridization. Our study here demonstrated these five species (Ph.
386
aureosulcata, Ph. bissetii, Ph. robustiramea, Ph. varioauriculata and Ph. nigra) should be
387
placed in Phyllostachys sect. Heterocladae with high support (100% MLBS) and their
388
relationships were resolved robustly. However, a broader taxonomic sampling is still
389
needed to confirm this result as we did not include all Phyllostachys species.
AC C
EP
TE D
M AN U
SC
RI PT
370
390 391
5.
Conclusions
ACCEPTED MANUSCRIPT In this study, we first explored the appropriate extraction and preservation method for
393
DNA materials using six representative woody bamboo species. We found that the
394
modified CTAB approach performed better in extracting high quality DNA than the
395
DNAsecure plant kit, and dry-powdered DNA had better preservation effect than TE-
396
dissolved DNA, RT-preserved-material and LT-preserved-material. Then we chose 21
397
bamboo species for MiddRAD sequencing to investigate impacts of DNA quality on this
398
technology. We found that DNA degradation to moderate level had little effect on
399
MiddRAD-seq. The reduced-representation sequencing technology was robust until total
400
genomic DNA was severely degraded. At last, we reconstructed the phylogeny of 20
401
bamboo species and found the Phyllostachys might be a monophyletic group and
402
demonstrated systematic positions of some disputable Phyllostachys species. As ddRAD
403
sequencing are becoming increasingly popular for phylogenetic studies, our study would
404
provide helpful suggestions for preserving molecular material of plants and application of
405
ddRAD-seq to reconstruct phylogeny of major plant taxa.
TE D
M AN U
SC
RI PT
392
408
Acknowledgements
AC C
407
EP
406
This work was supported by the National Natural Science Foundation of China
409
(Grant No. 31470322 and 31430011). We thank Dr. Li-Na Zhang, Dr. Xian-Zhi Zhang,
410
Dr. Wen-Cai Wang for providing DNA samples of some species and Ms. Jing-Xia Liu for
411
collecting leaf material of I. singulispicula 16001. We are grateful to Prof. Kai-Feng Hu,
412
Prof. Jun-Bo Yang, Ms. Jing Yang and Mr. Ji-Xiong Yang at Kunming Institute of
413
Botany,
CAS
for
providing
experimental
supports.
ACCEPTED MANUSCRIPT 414
References Akinlabi, E. T., Anane-Fenin, K., Akwada, D. R., 2017. Bamboo Taxonomy and
416
Distribution Across the Globe. Bamboo: The Multipurpose Plant. Akinlabi, E. T.,
417
Anane-Fenin, K., Akwada, D. R., Springer International Publishing: 1-37.
RI PT
415
Andrews, K. R., Good, J. M., Miller, M. R., Luikart, G., Hohenlohe, P. A., 2016.
419
Harnessing the power of RADseq for ecological and evolutionary genomics. Nat. Rev.
420
Genet. 17(2): 81-92.
422
Andrews, S., 2014. FastQC: A quality control tool for high throughput sequence data.
M AN U
421
SC
418
http://www.bioinformatics.babraham.ac.uk/projects/fastqc.
Baird, N. A., Etter, P. D., Atwood, T. S., Currey, M. C., Shiver, A. L., Lewis, Z. A.,
424
Selker, E. U., Cresko, W. A., Johnson, E. A., 2008. Rapid SNP discovery and genetic
425
mapping using sequenced RAD markers. PloS One 3(10).
428 429 430 431
the bamboos (Poaceae: Bambusoideae). Bamboo Sci. Cult. 24(1): 1-10. Cariou, M., Duret, L., Charlat, S., 2013. Is RAD-seq suitable for phylogenetic inference?
EP
427
Bamboo Phylogeny Group (BPG), 2012. An updated tribal and subtribal classification of
An in silico assessment and optimization. Ecol. Evol. 3(4): 846-852.
AC C
426
TE D
423
Catchen, J., Hohenlohe, P. A., Bassham, S., Amores, A., Cresko, W. A., 2013. Stacks: an analysis tool set for population genomics. Mol. Ecol. 22(11): 3124-3140.
432
Catchen, J. M., Amores, A., Hohenlohe, P., Cresko, W., Postlethwait, J. H., 2011. Stacks:
433
Building and Genotyping Loci De Novo From Short-Read Sequences. G3-Genes
434
Genom. Genet. 1(3): 171-182.
ACCEPTED MANUSCRIPT 435
Cruaud, A., Gautier, M., Galan, M., Foucaud, J., Sauné, L., Genson, G., Dubois, E.,
436
Nidelet, S., Deuve, T., Rasplus, J.-Y., 2014. Empirical assessment of RAD
437
sequencing for interspecific phylogeny. Mol. Biol. Evol. 31(5): 1272-1274.
441 442 443 444 445
RI PT
440
DNA extraction from hymenopterous insects. Insect Mol. Biol. 5(1): 21-24.
Doyle, J. J., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem bull 19: 11-15.
SC
439
Dillon, N., Austin, A. D., Bartowsky, E., 1996. Comparison of preservation techniques for
Doyle, J. J., Dickson, E. E., 1987. Preservation of Plant-Samples for DNA Restriction
M AN U
438
Endonuclease Analysis. Taxon 36(4): 715-722.
Friar, E., Kochert, G., 1991. Bamboo germplasm screening with nuclear restriction fragment length Polymorphisms. Theor. Appl. Genet. 82(6): 697-703. Graham, C. F., Glenn, T. C., McArthur, A. G., Boreham, D. R., Kieran, T., Lance, S.,
447
Manzon, R. G., Martino, J. A., Pierson, T., Rogers, S. M., 2015. Impacts of degraded
448
DNA on restriction enzyme associated DNA sequencing (RADSeq). Mol. Ecol.
449
Resour. 15: 1304-1315.
451 452
EP
Gray, M. A., Zoe, A. P., Kellogg, C. A., 2013. Comparison of DNA preservation methods
AC C
450
TE D
446
for environmental bacterial community samples. FEMS Microbiol. Ecol. 83(2): 468– 477.
453
Hodkinson, T. R., Renvoize, S. A., Chonghaile, G. N., Stapleton, C. M., Chase, M. W.,
454
2000. A Comparison of ITS Nuclear rDNA Sequence Data and AFLP Markers for
455
Phylogenetic Studies in Phyllostachys (Bambusoideae, Poaceae). J. Plant Res. 113(3):
456
259-269.
ACCEPTED MANUSCRIPT 457 458
Langmead, B., Salzberg, S. L., 2012. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9(4): 357. Ledoux, J. B., Aurelle, D., Féral, J. P., Garrabou, J., 2013. Molecular forensics in the
460
precious Mediterranean red coral, Corallium rubrum : testing DNA extraction and
461
microsatellite genotyping using dried colonies. Conserv. Genet. 5(2): 327-330.
RI PT
459
Li, D. Z., Wang, Z. P., Zhu, Z. D., Xia, N. H., Jia, L. Z., Guo, Z. H., Yang, G. Y.,
463
Stapleton, C. M. A., 2006. Flora of China Vol. 22 (Poaceae, Tribe Bambuseae).
464
Beijing, Science Press.
M AN U
SC
462
465
Liang, H., Fu, M., Yang, G., Chen, J., Yang, X., 2016. Effects of Different Preservation
466
Methods on Yield and Quality of Total Genomic DNA of Tamarix chinensis.
467
Genomics & Applied Biology 35(8): 2168-2172.
Maxine, P. P., Andrea, C. T., 2003. Extensive evaluation of faecal preservation and DNA
469
extraction methods in Australian native and introduced species. Aust. J. Zool. 51(4):
470
341-355.
TE D
468
Mitchell, K. R., Takacsvesbach, C. D., 2008. A comparison of methods for total
472
community DNA preservation and extraction from various thermal environments. J.
AC C
473
EP
471
Ind. Microbiol. Biot. 35(10): 1139.
474
Peterson, B. K., Weber, J. N., Kay, E. H., Fisher, H. S., Hoekstra, H. E., 2012. Double
475
digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in
476
model and non-model species. PloS One 7(5): e37135.
ACCEPTED MANUSCRIPT 477
Poland, J. A., Brown, P. J., Sorrells, M. E., Jannink, J.-L., 2012. Development of high-
478
density genetic maps for barley and wheat using a novel two-enzyme genotyping-by-
479
sequencing approach. PloS One 7(2): e32253.
482 483
RI PT
481
Prugh, L. R., Ritland, C. E., Arthur, S. M., Krebs, C. J., 2005. Monitoring coyote population dynamics by genotyping faeces. Mol. Ecol. 14(5): 1585–1596.
Puritz, J. B., Matz, M. V., Toonen, R. J., Weber, J. N., Bolnick, D. I., Bird, C. E., 2014. Demystifying the RAD fad. Mol. Ecol. 23(24): 5937-5942.
SC
480
Qin, H. T., Yang, G. Q., Provan, J., Liu, J., Gao, L. M., 2017. Using MiddRAD-seq data to
485
develop polymorphic microsatellite markers for an endangered yew species. Plant
486
Diversity 39(5): 294-299.
488
Rubin, B. E. R., Ree, R. H., Moreau, C. S., 2012. Inferring phylogenies from RAD sequence data. PLoS One 7(4): e33394.
TE D
487
M AN U
484
Scandura, M., Capitani, C., Iacolina, L., Marco, A., 2006. An empirical approach for
490
reliable microsatellite genotyping of wolf DNA from multiple noninvasive sources.
491
Conserv. Genet. 7(6): 1013-1013.
493
Stamatakis, A., 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis
AC C
492
EP
489
of large phylogenies. Bioinformatics 30(9): 1312-1313.
494
Suzuki, S., 1978. Index to Japanese Bambusaceae. Gakken Co. Ltd, Tokyo.
495
Takahashi, T., Nagata, N., Sota, T., 2014. Application of RAD-based phylogenetics to
496
complex relationships among variously related taxa in a species flock. Mol.
497
Phylogenet. Evol. 80: 137-144.
ACCEPTED MANUSCRIPT 498
Tin, M. M., Economo, E. P., Mikheyev, A. S., 2014. Sequencing degraded DNA from non-
499
destructively sampled museum specimens for RAD-tagging and low-coverage shotgun
500
phylogenetics. PLoS One 9(5): e96793. Tin, M. M. Y., Rheindt, F. E., Cros, E., Mikheyev, A. S., 2015. Degenerate adaptor
502
sequences for detecting PCR duplicates in reduced representation sequencing data
503
improve genotype calling accuracy. Mol. Ecol. Resour. 15(2): 329-336.
RI PT
501
Triplett, J. K., Clark, L. G., Fisher, A. E., Wen, J., 2014. Independent allopolyploidization
505
events preceded speciation in the temperate and tropical woody bamboos. New Phytol.
506
204(1): 66-73.
M AN U
SC
504
Wang, X. Q., Ye, X. Y., Zhao, L., Li, D. Z., Guo, Z. H., Zhuang, H. F., 2017. Genome-
508
wide RAD sequencing data provide unprecedented resolution of the phylogeny of
509
temperate bamboos (Poaceae: Bambusoideae). Sci. Rep-UK. 7.
TE D
507
Wang, X. Q., Zhao, L., Eaton, D. A. R., Li, D. Z., Guo, Z. H., 2013. Identification of SNP
511
markers for inferring phylogeny in temperate bamboos (Poaceae: Bambusoideae)
512
using RAD sequencing. Mol. Ecol. Resour. 13(5): 938-945.
514 515
Wysocki, W. P., Ruiz-Sanchez, E., Yin, Y., Duvall, M. R., 2016. The floral transcriptomes
AC C
513
EP
510
of four bamboo species (Bambusoideae; Poaceae): support for common ancestry among woody bamboos. BMC Genomics 17(1): 384.
516
Yang, G. Q., Chen, Y. M., Wang, J. P., Guo, C., Zhao, L., Wang, X. Y., Guo, Y., Li, L., Li,
517
D. Z., Guo, Z. H., 2016. Development of a universal and simplified ddRAD library
518
preparation approach for SNP discovery and genotyping in angiosperm plants. Plant
519
Methods 12.
ACCEPTED MANUSCRIPT Zhang, X. Z., Zeng, C. X., Ma, P. F., Haevermans, T., Zhang, Y. X., Zhang, L. N., Guo, Z.
521
H., Li, D. Z., 2016. Multi-locus plastid phylogenetic biogeography supports the Asian
522
hypothesis of the temperate woody bamboos (Poaceae: Bambusoideae). Mol.
523
Phylogenet. Evol. 96: 118-129.
RI PT
520
Zhang, Y. X., Zeng, C. X., Li, D. Z., 2012. Complex evolution in Arundinarieae (Poaceae:
525
Bambusoideae): Incongruence between plastid and nuclear GBSSI gene phylogenies.
526
Mol. Phylogenet. Evol. 63(3): 777-797.
SC
524
Zhao, H. S., Peng, Z. H., Fei, B. H., Li, L. B., Hu, T., Gao, Z. M., Jiang, Z. H., 2014.
528
BambooGDB: a bamboo genome database with functional annotation and an analysis
529
platform. Database -Oxford.
530
AC C
EP
TE D
531
M AN U
527
ACCEPTED MANUSCRIPT 532
Figure Legends
533
Fig .1 The whole experimental flowchart. Fresh leaves of each bamboo were divided into
534
three equal parts (Replicate1, Replicate2, and Replicate3). Replicate1 was dried with silica
535
gel at room temperature (RT), while Replicate2 was sealed within zip-lock bags and stored
536
in -80
537
different methods (the modified CTAB protocol and DNAsecure plant kit).
refrigerator (LW). Replicate3 was used for DNA extraction directly with two
538 Fig. 2 The electrophoresis result of initial total genomic DNA. M: Marker. A~F: DNA
540
extracted by modified CTAB procedure. G~L: DNA extracted by DNAsecure plant kit.
541
Species name corresponding to A-L are shown in Table 2.
542
RI PT
539
Fig. 3 The electrophoresis result of total genomic DNA extracted from leaves in different
544
preserve time using a modified CTAB procedure. (a) 6 months, (b) 12 months, (c) 24
545
months, (d) 36 months. M: Marker. Species name corresponding to A-L are shown in Table
546
2. Subscript R represents that DNA was extracted from leaves stored in silica gel on RT.
547
Subscript L represents that DNA was extracted from leaves stored in -80
SC
543
M AN U
548
refrigerator.
549
Fig. 4 The electrophoresis result of total genomic DNA extracted from leaves in different
550
preserve time using DNAsecure plant kit. (a) 6 months, (b) 12 months, (c) 24 months, (d)
551
36 months. M: Marker. Species name corresponding to A-L are shown in Table 2.
552
Subscript R represents that DNA was extracted from leaves stored in silica gel on RT.
553
Subscript L represents that DNA was extracted from leaves stored in -80
554
refrigerator.
Fig. 5 The electrophoresis result of total genomic DNA dissolved in TE after different
556
preserve time. (a) 6 months, (b) 12 months, (c) 24 months, (d) 36 months. M: Marker.
557
Species name corresponding to A-L are shown in Table 2.
TE D
555
558
Fig. 6 The electrophoresis result of dry powdered DNA in different preserve time. (a) 6
560
months, (b) 12 months, (c) 36 months. M: Marker. Species name corresponding to A-L are
561
shown in Table 2.
562
EP
559
Fig. 7 The electrophoresis result of DNA in 21 individuals. M: Marker. 1: DNA of I.
564
singulispicula 12162, 2-3: DNA were extracted with fresh materials, 4-15: DNA were
565
dissolved in TE and stored in -80
566
gel stored leaf materials for 36 months. Species name that correspond to the numbers are
567
shown in Table 1.
568 569
AC C
563
for 36 months, 16-21: DNA were extracted from silica
Fig. 8 MiddRAD-seq data summaries for two individuals of I. singulispicula. This includes
570
raw reads and clean reads number (a), Unexpected enzyme cutting sites ratio (b), Mapping
571
ratio (c), Tags number (d), data quality of I. singulispicula 16001 read1 (e) and I.
572
singulispicula 12162 read1 (f).
573
ACCEPTED MANUSCRIPT 574
Fig. 9 Maximum likelihood phylogenetic reconstruction of twenty bamboo species. Two
575
subclades were recognized in Phyllostachys, Phyllostachys sect. Phyllostachys (subclade
576
1), and Phyllostachys sect. Heterocladae (subclade 2).
577
AC C
EP
TE D
M AN U
SC
RI PT
578
ACCEPTED MANUSCRIPT 579
Table 1 Sample location and storage information. Taxon Location
No. in Fig. 7
Storage method
Storage time(month)
Xishuangbanna, Yunnan, China
1
M1
48
Indosasa singulispicula 16001
Xishuangbanna, Yunnan, China
2
M
0
Phyllostachys nigra
KIB, Yunnan, China 3
3
M
0
Pleioblastus chino
Kew, UK 4
4
D2
36
Sasa bitchuensis
Kew, UK
5
D
36
Sasa ramosa
Kew, UK
6
D
36
Semiarundinaria fortis
Japan
7
D
36
Phyllostachys robustiramea
Anji, Zhejiang, China
8
D
36
Phyllostachys rubromarginata
Anji, Zhejiang, China
9
D
36
Phyllostachys kwangsiensis
Anji, Zhejiang, China
10
D
36
Phyllostachys mannii
Anji, Zhejiang, China
11
D
36
Phyllostachys sulphurea var. viridis
Anji, Zhejiang, China
12
D
36
Phyllostachys heteroclada
Anji, Zhejiang, China
13
D
36
Phyllostachys vivax
Guangde, Anhui, China
14
D
36
Phyllostachys varioauriculata
Guangde, Anhui, China
15
D
36
Anji, Zhejiang, China
16
M
36
Anji, Zhejiang, China
17
M
36
Anji, Zhejiang, China
18
M
36
Phyllostachys rubicunda
SC
M AN U
EP
Phyllostachys prominens
TE D
Phyllostachys nidularia
RI PT
Indosasa singulispicula 12161
Phyllostachys aureosulcata
Anji, Zhejiang, China
19
M
36
Phyllostachys acuta
Guangde, Anhui, China
20
M
36
Guangde, Anhui, China
21
M
36
AC C
Phyllostachys bissetii 580
1
581
solution. 3 KIB represents Kunming Institute of Botany, Chinese Academy of Sciences,
582
4
583
M represents leaf material stored in silica gel, while 2 D represents DNA dissolved in TE KEW represents Royal Botanic Gardens in the UK.
ACCEPTED MANUSCRIPT Table 2 Concentration and purity of initial total genomic DNA .
Modified CTAB procedure
Species ID
Concentration (ng/ul)
A260/280
Dendrocalamus latiflorus 1
A
1263.5
2.0
Bambusa multiplex cv. AlphonseKarr 1
B
824.4
1.99
Bambusa emeiensis 1
C
940.4
1.99
Indosasa hispida cv. rainbow 2
D
647.7
1.99
Phyllostachys edulis 2
E
766.6
2.0
Acidosasa purpurea 2
F
1021.9
2.0
Dendrocalamus latiflorus
G
217.9
1.94
119
1.83
I
136.5
1.95
Indosasa hispida cv. rainbow
J
167.4
1.89
Phyllostachys edulis
K
207.7
1.85
L
171.6
1.89
Species
DNAsecure plant kit
Bambusa emeiensis
Acidosasa purpurea
TE D
587
tropical woody bamboos, 2 temperate woody bamboos.
EP
586
1
AC C
585
H
M AN U
Bambusa multiplex cv. AlphonseKarr
RI PT
Extraction method
SC
584
ACCEPTED MANUSCRIPT Table 3 MiddRAD-seq data summary of 20 selected species. Number of tags
Alignment ratio (%)
Unexcepted enzyme cutting site ratio (%)
Missing ratio (%)
Indosasa singulispicula 1
1.68
175588
67
10.84
32.46
Phyllostachys nigra
6.36
199113
81.8
8.43
36.02
Pleioblastus chino
1.74
165174
64.74
7.9
34.03
Sasa bitchuensis
2.89
154681
66.43
7.16
34.65
Sasa ramosa
2.23
158035
66.7
6.6
39.27
Semiarundinaria fortis
2.87
176716
65.56
8.24
34.17
Phyllostachys robustiramea
2.66
188296
81.18
4.68
24.24
Phyllostachys kwangsiensis
2.65
176921
86.48
5.11
26.91
Phyllostachys rubromarginata
2.31
79.79
4.53
24.24
Phyllostachys mannii
3.47
79.74
7.06
22.78
Phyllostachys sulphurea var. viridis
2.58
81.98
7.12
20.52
Phyllostachys heteroclada
4.4
204362
78.34
5.1
22.33
Phyllostachys vivax
3.63
191962
74.53
7.2
19.04
78.38
8.51
15.25
Phyllostachys varioauriculata
1.28
M AN U 196504
164771
154792
3.71
207817
79.77
6.03
11.01
3.76
191329
78.25
4.71
22.4
3.56
195164
81.44
4.8
16.45
1.9
188183
77.33
7.04
17.18
Phyllostachys bissetii
1.88
183433
79.46
7.68
13.77
Phyllostachys rubicunda
3.54
187912
78.37
7.01
21.42
Phyllostachys nidularia Phyllostachys prominens
589 590
1
AC C
Phyllostachys acuta
EP
Phyllostachys aureosulcata
192009
TE D
Taxon
SC
Data size(Gb)
RI PT
588
Indosasa singulispicula represents Indosasa singulispicula 16001 in Table 1.
ACCEPTED MANUSCRIPT
106754
1
62.66
p6
706221
75595
1
59.03
p7
526555
53073
1
55.28
p8
372902
36575
0
51.28
p9
250782
25252
4
p10
160448
17184
6
p11
99010
11745
6
p12
59843
8109
2
p13
35754
5589
1
p14
21063
3821
0
p15
11904
2550
RI PT
914416
47.03 42.54
M AN U
SC
37.92
7
33.28 28.76 24.41 20.1
P value, minimum number of individuals a locus must be present in to process a locus.
TE D
1
p5
EP
592
Table 4 Statistics for different data sets used in phylogenetic analyses. No. of No. of Nodes P value 1 Average missing ratio (%) SNPs Loci (MLBS