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Apr 17, 2018 - d in TE extracted by modifie d CTAB procedure extracted by. DNAsecure plant kit. Dry powder ed DNA extracted by modifie d CTAB procedure.

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

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A comparison of different methods for preserving plant

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molecular materials and impacts of degraded DNA on ddRAD

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sequencing

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Ying Guo a,b , Guo-Qian Yang a,b , Yun-Mei Chen a,b , De-Zhu Li a* , and Zhen-Hua

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Guo a*

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a

Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of

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Sciences, Kunming 650201, China

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b

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*Author for correspondence.

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Kunming Institute of Botany, Chinese Academy of Sciences

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132 Lanhei Road, Kunming, Yunnan 650201, China

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E-mail: [email protected] , [email protected]

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Tel: +86-871-65223503

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Fax: +86-871-65223153

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University of Chinese Academy of Sciences, Beijing 100049, China

ACCEPTED MANUSCRIPT Abstract Getting well-conditioned plant materials for multitudinous experiments is

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difficult in many research projects, so exploring how to preserve materials is of special

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importance after they were collected in case that biological macromolecules like DNA to

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be degraded. Although some researches have demonstrated that DNA degradation has little

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effect on some traditional molecular markers, the impacts of DNA degradation on ddRAD-

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seq, a popular reduced-representation sequencing technology, have not been adequately

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investigated. In this study, we first chose six woody bamboo species (Bambusoideae,

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Poaceae) to explore appropriate methods for preserving molecular materials with two DNA

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extraction approaches. Then we sequenced another twenty-one bamboos and examined the

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impacts of DNA quality on data generation using ddRAD-seq technique (MiddRAD-seq).

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Finally, we reconstructed phylogenies of twenty woody bamboo species. We found dry-

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powdered DNA had the longest preserving time compared with TE-dissolved DNA which

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were extracted with both modified CTAB protocol and DNAsecure plant kit. ddRAD-seq

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was robust except that DNA was severely degraded and we also demonstrated the

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systematic positions of the sampled Phyllostachys species. Our results suggest that dry-

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powdered DNA is a commendable way to preserve molecular materials. Furthermore, DNA

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degradation to moderate level has little effect on reduced representation sequencing

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techniques represented by ddRAD-seq.

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Keywords molecular materials; DNA extraction; DNA preservation; DNA quality;

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ddRAD-seq; bamboo; phylogeny

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1.

Introduction Collecting experimental materials is the necessary prerequisite for almost every

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biological research project. It’s common that researchers collect plant materials from

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different locations or even from different countries in many cases. As some plants are

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endemic species which are difficult to get samples, it’s of crucial importance to preserve

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their biological samples in an appropriate way once obtained (Doyle and Dickson, 1987).

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DNA extracted from plant materials is often degraded to varying degrees and it may be

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adverse to future researches. However, few researches lay stress on this problem and

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most of these researches were designed for one species or different varieties with a short

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preserving time ranging from few hours to few months, so the conclusions drawn were

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not very convictive, especially for some plants that degraded slightly in a short time

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(Liang et al., 2016).

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Degraded genomic DNA may have little effect on some traditional molecular

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markers, such as microsatellites (Ledoux et al., 2013; Prugh et al., 2005; Qin et al., 2017;

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Scandura et al., 2006). However, with the application of next-generation DNA

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sequencing (NGS), more and more reduced representation sequencing methods were used

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in recent molecular researches, such as molecular phylogenetics and molecular ecology

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(Baird et al., 2008; Peterson et al., 2012; Poland et al., 2012; Yang et al., 2016).

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Restriction-site associated DNA sequencing (RAD-seq) was first introduced in 2008 as a

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rapid SNP discovery and genotyping method (Baird et al., 2008). This technology utilizes

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RAD-tags which are short DNA fragments adjacent to a particular restriction enzyme

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recognition site to reflect the sequence characteristics of the whole genome and construct

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ACCEPTED MANUSCRIPT sequencing library for high-throughput sequencing (Baird et al., 2008; Peterson et al.,

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2012). Although it is originally designed for intraspecific genomic analysis, many recent

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studies which involve in many fields, such as phylogenetic analysis, population genomics,

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ecological and evolutionary genomics, have proved that it can also be useful at

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interspecific levels (Andrews et al., 2016; Cariou et al., 2013; Cruaud et al., 2014; Rubin

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et al., 2012; Takahashi et al., 2014; Wang et al., 2013). Nevertheless, as a relatively new

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technology, there are few researches about the impacts of DNA degradation on this

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technology. Tin et al (2014) and Graham et al (2015) used few animal specimens or fresh

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tissues (ants and whitefish) to investigate this problem respectively. However, as the

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research objects of these two studies are animals, it is necessary to fill this gap in plants

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because of the essential difference between animals and plants materials.

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The Bambusoideae (bamboos), including more than 1400 described species in nearly

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120 genera, is one of the most important member of Poaceae (Akinlabi et al., 2017; BPG,

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2012), and has great ecological and economic value as it provides food and raw materials

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for construction and manufacturing, especially woody bamboos (Li et al., 2006).

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Bambusoideae is also a difficult group in taxonomy as their complex polyploidy

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evolutionary history and slow evolutionary rate (Triplett et al., 2014; Zhang et al., 2012).

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Although Bambusoideae has been extensively studied in evolutionary genetics contexts, its

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phylogenetic relationship is still difficult to resolve (Wysocki et al., 2016; Zhang et al.,

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2016). In recent years, analyses based on these RAD tags provide an opportunity to yield

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robust phylogenetic inferences on bamboo phylogenetics (Wang et al., 2017; Wang et al.,

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2013). The woody bamboos are widely distributed around the world, from Asia, America

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ACCEPTED MANUSCRIPT to Africa (Akinlabi et al., 2017; Bamboo, 2012). Therefore, it is a good model for

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conducting researches on plant material preservation. Here, we applied two DNA

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extraction methods and four different preservation methods on 3 temperate woody

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bamboos and 3 tropical woody bamboos to explore the appropriate preservation method for

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plant materials monitored as longest up to three years. Besides, we used the modified

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ddRAD-seq (MiddRAD) with 21 woody bamboos to examine the effect of DNA quality on

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RAD-seq. Our main goals were: (1) to find an appropriate preservation method for

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precious plant materials in order to obtain high quality DNA for subsequent DNA analysis,

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and (2) to examine the impact of degraded DNA on ddRAD-seq approach (MiddRAD-seq)

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using the STACKS bioinformatics pipeline (Catchen et al., 2013; Yang et al., 2016).

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Materials and Methods

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2.1 Plant Materials and Treatments

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Six bamboo species including three temperate woody bamboos (Phyllostachys edulis,

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Indosasa hispida cv. rainbow, Acidosasa purpurea) and three tropical woody bamboos

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(Dendrocalamus latiflorus, Bambusa multiplex cv. Alphonse-Karr, B. emeiensis) were

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chosen to explore the ideal plant leaf material preservation method with observing time

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as long as three years. Fresh leaves from each bamboo species were collected and divided

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into three equal parts (Replicate1, Replicate2, and Replicate3). Replicate1 was dried with

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silica gel at room temperature (RT), while Replicate2 was sealed within zip-lock bags

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and stored in -80

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two different methods (the modified CTAB protocol and DNAsecure plant kit)

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refrigerator. Replicate3 was used for DNA extraction directly with

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respectively. Then total genomic DNA extracted from Replicate3 was divided equally

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into ten tubes, of which five of them were dissolved in TE solution and another five tubes

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were dried by a freeze drier to get dry-powdered DNA. Finally, all ten tubes were stored

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in -80

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material which stored in low temperature (LT, Replicate1) and RT (Replicate2) were

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extracted and detected with electrophoresis and Nanodrop respectively. After a year of

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observation, we found that there was little difference between detection results of six-

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months storage and twelve-months storage. So we extended the detection period to every

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12 months. Considering the possible longer preserving time of dry-powdered DNA, we

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extended the detection period of it to 24 months. If the DNA was detected degraded, this

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sample was re-extracted and detected again to confirm the degradation. The whole

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experimental flowchart is shown in Fig. 1. Fresh leaf materials of these six species were

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all collected from plants grown in Kunming Institute of Botany, Chinese Academy of

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Sciences (KIBCAS) (N25°07′04.9″, E102°44′15.2″).

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refrigerator. Every six months later, DNA from Replicate3 were detected and the

Furthermore, we sequenced 21 temperate woody bamboos (I. singulispicula has two

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individuals) which were collected from different locations with ddRAD-seq (Yang et al.,

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2016) to examine the impact of DNA quality on RAD-seq (Table 1). DNA of some

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species were extracted immediately with fresh samples, some were extracted after

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different preserving time, detailed information was shown in Table 1.

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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

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(Doyle, 1987). Specific steps are as follows. Firstly, the mortars were washed and dried

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before starting the experiment; then the mortars were burned to sterilize using alcohol;

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after which, 20 mg samples with moderate quartz sand were put in liquid nitrogen and

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grounded quickly; then the grounded powder were transferred into a 2ml clean

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microcentrifuge tube and were mixed immediately with 1ml of 4×CTAB extracting

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solution to which 1% of β-mercaptoethanol (BME) had been added; then the samples

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were placed in the 65

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added, mixed and centrifuged at 9000 rpm when the tube was cooled to room temperature,

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then the supernatant was transferred in another 2ml clean microcentrifuge tube and this

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step was repeated once again; then the supernatant was mixed with 0.7 volumes of

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isopropanol and the solution was incubated in -20

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centrifuged at 10000 rpm for 8 minutes, the supernatant liquor was discarded and the

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DNA was cleaned with 70% and absolute ethyl alcohol twice respectively, then dry

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powdered DNA was produced by putting the tube in vacuum centrifuge concentrator at

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50

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and incubated the mixture with 0.5ul RNase at 37

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solution was stored in -80 .

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water for 1h; 1ml of chloroform-isoamyl alcohol (24:1) was

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for 1h; after that, the solution were

for 3-5 minutes; after that, we dissolved the dry-powdered DNA in 50ul TE solution

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for 1.5-2h to digest RNA; finally, the

The integrality of total genomic DNA was detected by agarose gel electrophoresis

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while

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spectrophotometer (Thermo Fisher Scientific, Delaware, USA).

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concentration

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purity

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DNA

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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

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concentration of total genomic DNA and diluted DNA to the proper concentration (40

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ng/ul). Because of the different preserving time and methods, the samples were

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sequenced in different sequencing batches. ddRAD libraries were prepared according to

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Yang et al (2016). Each sample was digested with two enzymes, i.e. Ava

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600-700 base pair (bp) DNA fragments were selected from agarose gel and recovered by

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E.Z.N.A DNA gel extraction kit (D2500-02). We sequenced all ddRAD libraries on the

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Illumina HiSeq X10 (Illumina, San Diego, CA, USA) by employing paired-end 150bp

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sequencing mode at the Cloud Health Genomics Company (Shanghai, China).

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2.4 Data analysis

Clean data were obtained after two processing steps. Firstly, raw data were de-

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multiplexed by process_radtags program implemented in STACKS version 1.41 (Catchen

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et al., 2013; Catchen et al., 2011) and the sequence quality of each sample was checked

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using FastQC version 0.11.2 (Andrews, 2014). Then, adapter reads and low-quality bases

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which were below a Phred score of Q10 were deleted and the sequences were truncated to

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a final length of 140bp with the process_radtags program. After reads trimming, ustacks

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program was used to merge short-read sequences into tags/loci with ranging settings for

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minimum depth of coverage (m = 5~15) and a maximum of 5-bp difference allowed

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between stacks (M = 5). Then cstacks program was used to merge loci into catalog with

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fourteen mismatches allowed between sample loci (n = 14). The sstacks program was

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applied to match loci from an individual against the catalog built by cstacks and loci that

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matched more than one catalog locus were excluded. Finally, the populations program

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ACCEPTED MANUSCRIPT was used to output single nucleotide polymorphism markers (SNPs) in phylip format.

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After that, we used custom shell commands to compute the RAD tags number and

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unexpected enzyme cutting site ratio for each sample. To determine and compare the

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mapping ratio of reads to the genome, clean data of each individual was mapped to Ph.

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edulis genome scaffolds (Zhao et al., 2014) with Bowtie 2.2.9 respectively (Langmead

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and Salzberg, 2012).

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Finally, the data set was analyzed with a maximum-likelihood method using the

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general time-reversible (GTR) model, which was implemented in RAxML-HPC BlackBox

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version 8.2.10 on the CIPRES Science Gateway web server, with a rapid bootstrapping

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analysis of 1000 bootstrap replicates (Stamatakis, 2014).

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Results

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3.1 Exploring appropriate methods for preserving DNA materials

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3.1.1 Initial DNA quality detection

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Initial total genomic DNA was extracted from fresh leaves of six woody bamboos

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using two methods and detected by agarose gel electrophoresis and spectrophotometer

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respectively. The electrophoresis result showed that total genomic DNA extracted by

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modified CTAB method all had clear main bands (Fig. 2A-F), while slight degradations

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were found for DNA extracted by DNAsecure plant kit (Fig. 2G-L). The absorption ratio

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of DNA at 260nm and 280nm were all between 1.8 and 2.0 while the DNA concentration

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of samples extracted by modified CTAB method was higher than DNA extracted by

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DNAsecure plant kit (Table 2).

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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

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materials stored under room temperature (RT, in silica gel) and low temperature (LT, fresh

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leaves) for 6months, 12 months, 24 months and 36 months respectively. The

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electrophoresis results showed that DNA in 6 months all had clear main band with no

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obvious degradation or slight degradation (Fig. 3a), DNA degradation increased in 12

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months for RT-stored materials and LT-stored materials (Fig. 3b), while some individuals

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showed unclear main DNA bands and obvious degradations after 24 months storing (Fig.

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3c, A L -F L ). Two individuals had degraded completely after 36 months storage, with one

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(Fig. 3d, A R ) extracted from RT leaf material and the other one (Fig. 3d, B L ) extracted

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from LT leaf material.

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Secondly, total genomic DNA was extracted with DNAsecure plant kit from leaf

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materials stored under RT (in silica gel) and LT (fresh leaves) after storing 6 months, 12

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months, 24 months and 36 months respectively. The electrophoresis results showed that

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DNA in 6 months all had clear main band with slight degradation for LT-stored materials

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(Fig. 4a). DNA degradation increased in 12 months for RT-stored materials and LT-stored

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materials (Fig. 4b). Moderate DNA degradation for most RT-stored materials and LT-

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stored materials were found after 24 months of storage (Fig. 4c), of which three

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individuals had ambiguous main DNA bands (Fig. 4c, G R , I R , J R ). One individual has

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degraded completely (Fig. 4d, G R ) and many individuals have unclear bands (degraded

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moderately) after 36 months. On the other hand, the brightness of DNA bands in Fig. 4

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was lower than that in Fig. 3 under the same condition, which might indicate the CTAB

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method is more efficient than the DNAsecure plant kit method in improving DNA

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concentration. Thirdly, total genomic DNA extracted from fresh bamboo leaves which were stored in

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TE solution was detected after being preserved for 6 months, 12 months, 24 months and 36

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months respectively. The electrophoresis results showed that DNA main bands were clear,

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bright and only slight degradations were found after storing 6 months and 12 months

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respectively (Fig. 5a, b). degradation increased in 24 months for DNA extracted by

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modified CTAB method and the DNAsecure plant kit (Fig. 5c). Furthermore, four

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individuals had ambiguous main bands and obvious degradations after 36 months storing

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and total genomic DNA of these four individuals were all extracted by modified CTAB

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method (Fig. 5d, C-F).

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was detected after 6 months, 12

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months and 36 months respectively. The electrophoresis result showed that main DNA

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bands were clear, bright and only slight degradations were found after 6-36 months of

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storage (Fig. 6). As the image of 36 months was taken with a different UV-

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spectrophotometer, this might bring difference to the brightness of images even under the

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same exposure rate. Even though this image was darker than other two images (Fig. 6a, b),

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we could learn the high integrity of 36-months-preserved DNA by comparing it to the

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DNA Marker. Nanodrop spectrophotometer detection also verified the high concentration

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and purity of total genomic DNA at different preserving time (Appendix: Table A).

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3.2 DNA quality evaluation through ddRAD sequence analysis

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ACCEPTED MANUSCRIPT To determine the quality of DNA preserved under different preserving time, we

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sequenced and analyzed two I. singulispicula individuals with MiddRAD-seq (Table 1).

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One (I. singulispicula 12162) was collected from Xishuangbanna, Yunnan province in

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2012 (N21°48′51.18″E101°22′51.12″, elevation 568 m), while the other (I. singulispicula

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16001) was collected from the same place in 2016. The electrophoresis result showed that

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DNA extracted from leaf of I. singulispicula 12162 was completely degraded while the

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other individual had clear band with only slight degradation (Fig. 7, No.1-2). We

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estimated the data quality of these two individuals using FastQC software and shell

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commands. The results showed the average reads quality of I. singulispicula 12162 had

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no obvious difference with I. singulispicula 16001, but the raw reads number, clean reads

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number and tags number of I. singulispicula 16001 were ten times larger than I.

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singulispicula 12162 (Fig. 8a, d, e, f). Moreover, there were more reads which had

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unexpected restriction enzyme cutting site and less reads which could be mapped to

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Phyllostachys edulis reference genome in I. singulispicula 12162 than the other

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individual (I. singulispicula 16001) (Fig. 8b, c). In summary, the data quality of I.

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singulispicula 12162 was clearly worse than I. singulispicula 16001 and it was supposed

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to be unfit for subsequent phylogenetic analyses.

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Besides, we evaluated data quality of twenty more bamboo species from different

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batches of ddRAD sequencing runs (Table 3). Among them, two species were extracted

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with fresh materials immediately and sequenced within one month after sample collection

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(Table 1, No. 2-3), twelve species were sequenced after their DNA dissolved in TE and

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stored in -80

for 36 months (Table 1, No. 4-15) and another six species were sequenced

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after their leaves stored in silica gel for 36 months (Table 1, No. 16-21). The

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electrophoresis result showed that DNA extracted from fresh leaves (Fig. 7, No. 2-3)

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were nearly intact, while DNA dissolved in TE and stored in -80

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slight degradation and DNA extracted from silica gel stored leaf materials (Fig. 7, No.

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16-21) had moderate degradation. Meanwhile, all of them had clear main DNA bands. We

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found the data of these twenty species were all of high quality. The data size of them was

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ranged from 1.28 G to 6.36 G (Table 3). Tags number was all between 150000 and

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210000, unexpected enzyme cutting site ratio of most species (except I. singulispicula)

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were below 10% (Table 3). As expected, alignment ratios of species in genus

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Phyllostachys to the reference genome were higher than that of species in other genus

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and data missing ratio were usually lower than that of other species (except Ph. nigra)

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(Table 3).

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(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

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of temperate woody bamboos with the Stacks software. Eleven data sets, ranging from

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11904 SNPs (p=15) to 914416 SNPs (p=5), were yielded and used for phylogenetic

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analyses with the maximum likelihood method. Topologies of phylogenetic trees which

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constructed from different data sets were largely congruent except low MLBS (Maximum

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Likelihood Bootstrap) of some nodes (Table 4). Phylogenetic analysis using 21063 SNPs

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data set (p=14) revealed robust support for the relationships between twenty species

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(100% MLBS, Fig. 9). Two clades were found. The first contained five species, of which

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four of them were native to Japan (Suzuki, 1978) and clustered together, sister to I.

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singulispicula. Within the first clade, Sasa bitchuensis was sister to S. ramose, forming a

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clade

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Semiarundinaria fortis. All the Phyllostachys members we used in this study were

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contained in the second clade with high support (100% MLBS), which agreed well with

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the phylogeny reported by Wang et al (2017). Two subclades were recognized in this

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clade: the first one contained six species which all belonged to Phyllostachys sect.

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Phyllostachys, while the second subclade (Phyllostachys sect. Heterocladae) contained

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another nine species, which largely agreed with the morphology-based taxonomy (Fig. 9).

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Notably,

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aureosulcata, Ph. bissetii) which had ambiguous systematic positions before were all

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clearly resolved in this study with 100% MLBS.

is

sister

to

another

clade

which

contains

Pleioblastus

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five

species

(Ph.

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nigra,

Ph.

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Ph.

varioauriculata,

Ph.

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4.

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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,

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which has a strong absorption effect on the ultraviolet ray. 230nm, 260nm and 280nm is

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maximum absorption peak of carbohydrate, nucleic acid, protein and phenols respectively.

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The ratio between them can be used to evaluate the purity of nucleic acid samples. The

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absorption ratio of 260nm to 280nm (A260/280) indicates high quality of DNA when it is

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between 1.8 and 2.0. When the ratio is higher than 2.0, it indicates that there is RNA

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pollution in the extracted DNA, while if it is lower than 1.8, the samples might contain

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small molecular pollutions such as protein or phenolic substances (Liang et al., 2016).

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From the electrophoresis and spectrophotometer results, we could learn the initial total

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genomic DNA extracted from fresh leaves by two DNA extraction methods was of high

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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

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12 months for LT material. The difference between DNA extracted from RT material and

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LT material after 36 months storing was indistinctive (Fig. 3, Fig. 4), which indicate that

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preserving leaf materials in silica gel or cryopreservation is not an efficient method to

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protect DNA from being degraded after a long-term storage time. This result is out of our

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expectation and we suppose it is probably due to complex secondary metabolites in plant

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leaves. The difference between these two preserving methods would be evident after a

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longer storage time. A comparison of the preserved effect of dry-powdered DNA and TE-

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dissolved DNA (Fig. 5, Fig. 6) revealed that the former had only slight degradation after

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36 months storing while the latter had obvious degradation after the same storage time,

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which means dry-powdered DNA could be preserved longer than TE-dissolved DNA, and

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dry-powdered DNA could be preserved at least 36 months without severe degradation. A

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comparison of the two extracting methods indicates that the purity of DNA extracted by

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two methods was both good, but the concentration of DNA extracted by the modified

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CTAB procedure is clearly higher than the other method (Table 2, Table A). However, we

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found that the DNA dissolved in TE solution of four individuals which had obvious

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degradation after 36 months storage were all extracted by the modified CTAB method (Fig.

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5). We detected them again to exclude the possible error of operation. This phenomenon

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ACCEPTED MANUSCRIPT might indicate that DNA extracted by CTAB method had shorter preserving time than the

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DNAsecure plant kit method, but it still need to be confirmed with longer observation time.

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Many previous researches were performed on animals or bacteria using one or few

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species with a short observing time (Dillon et al., 1996; Gray et al., 2013; Maxine and

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Andrea, 2003; Mitchell and Takacsvesbach, 2008). Compared to them, our study adopts

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more plant species and a more detailed experimental design and longer observation time,

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so our results could provide useful suggestions for plant materials preservation. We could

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conclude that: (1) The CTAB procedure is an appropriate method for extracting DNA from

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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

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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

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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

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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.

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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.

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Acknowledgements

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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

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Andrews, S., 2014. FastQC: A quality control tool for high throughput sequence data.

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Catchen, J. M., Amores, A., Hohenlohe, P., Cresko, W., Postlethwait, J. H., 2011. Stacks:

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develop polymorphic microsatellite markers for an endangered yew species. Plant

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Scandura, M., Capitani, C., Iacolina, L., Marco, A., 2006. An empirical approach for

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reliable microsatellite genotyping of wolf DNA from multiple noninvasive sources.

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Stamatakis, A., 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis

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Takahashi, T., Nagata, N., Sota, T., 2014. Application of RAD-based phylogenetics to

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complex relationships among variously related taxa in a species flock. Mol.

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Tin, M. M., Economo, E. P., Mikheyev, A. S., 2014. Sequencing degraded DNA from non-

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destructively sampled museum specimens for RAD-tagging and low-coverage shotgun

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Wysocki, W. P., Ruiz-Sanchez, E., Yin, Y., Duvall, M. R., 2016. The floral transcriptomes

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Yang, G. Q., Chen, Y. M., Wang, J. P., Guo, C., Zhao, L., Wang, X. Y., Guo, Y., Li, L., Li,

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D. Z., Guo, Z. H., 2016. Development of a universal and simplified ddRAD library

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preparation approach for SNP discovery and genotyping in angiosperm plants. Plant

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H., Li, D. Z., 2016. Multi-locus plastid phylogenetic biogeography supports the Asian

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Zhang, Y. X., Zeng, C. X., Li, D. Z., 2012. Complex evolution in Arundinarieae (Poaceae:

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Bambusoideae): Incongruence between plastid and nuclear GBSSI gene phylogenies.

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Mol. Phylogenet. Evol. 63(3): 777-797.

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Zhao, H. S., Peng, Z. H., Fei, B. H., Li, L. B., Hu, T., Gao, Z. M., Jiang, Z. H., 2014.

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BambooGDB: a bamboo genome database with functional annotation and an analysis

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platform. Database -Oxford.

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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

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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

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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.

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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

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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

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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

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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

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