ГЕНЕТИКА, 2014, том 50, № 3, с. 306-313
ГЕНЕТИКА РАСТЕНИЙ
УДК 575.1:582.542.1
Evaluation of Bamboo Genetic Diversity Using Morphological and SRAP Analyses
© 2014 S. Zhu, T. Liu, Q. Tang, L. Fu, Sh. Tang
Institute of Bast Fiber Crops and Center of Southern Economic Crops, Chinese Academy of Agricultural Sciences, Changsha, 410205 China e-mail: csec2012@163.com Received June 19, 2013
Bamboo is an important member of the giant grass subfamily Bambusoideae of Poaceae. In this study, 13 bamboo accessions belonging to 5 different genera were subjected to morphological evaluation and sequence-related amplified polymorphism (SRAP) analysis. Unweighted pair-group method of arithmetic averages (UPGMA) cluster analysis was used to construct a dendrogram and to estimate the genetic distances among accessions. On the basis of morphological characteristics, the 13 accessions were distinctly classified into 2 major clusters; 3 varieties, PPYX, PGNK, and PLYY were grouped as cluster A, and 10 accessions were categorized under cluster B. Similarity coefficients ranging from 0.23 to 0.96 indicated abundant genetic variation among bamboo varieties. Approximately 38 SRAP primer combinations generated 186 bands, with 150 bands (80.65%) showing polymorphisms among the 13 accessions. Based on SRAP analysis, 13 bamboo accessions were grouped into 3 major clusters. Five species comprised Cluster I (PASL, PLYY, PTSC, SCNK, and BMAK), which belongs to genus Phyllostachys. Cluster II consisted of 5 varieties, PASL, PLYY, PTSC, SCNK, and BMAK; Cluster III included 3 varieties, PGNK, PLSY, and BMRS. Comparison of the results generated by morphological and SRAP analyses showed that the classification based on SRAP markers was more concordant to the taxonomic results of Gamble than that performed using morphological characters, thus suggesting that SRAP analysis is more efficient in evaluating genetic diversity in bamboos compared to morphological analysis. The SRAP technique serves as an alternative method in assessing genetic diversity within bamboo collections.
DOI: 10.7868/S0016675814030138
Bamboo is an important member of the giant grass subfamily Bambusoideae of Poaceae and is distributed in various tropical and subtropical regions around the world. Bamboos play an important role in rural communities; tribes use bamboos in numerous ways, including house construction, agriculture, and human and animal nutrition [1]. The bamboo subfamily includes approximately 70 genera and 1,200 species, which are distributed worldwide [2]. Vegetative characteristics are generally used for classification of bamboos because they rarely produce flowers. Previous reports have shown that it may take as long as 120 years for a bamboo plant to bloom [3]. Certain bamboo species are known to never produce flowers [3, 4]. Taxono-mists thus rely solely on vegetative characteristics such as culm sheath and ligule for classification. However, vegetative characteristics are often influenced by environmental factors [5], thus rendering bamboo classification quite challenging. Alternatively, molecular markers can help in acquiring more accurate results for analyzing genetic diversity in plants. Previous studies on bamboo have utilized molecular markers generated from random amplified polymorphism DNA (RAPD), expressed sequence tag derived-simple sequence repeat (EST-SSR), simple sequence repeat (SSR), amplified fragment length polymorphisms (AFLPs) and restriction fragment length polymorphisms (RFLPs) analyses to examine genetic variabil-
ity [6—10]. The sequence-related amplified polymorphism (SRAP) technique is a simple and efficient DNA-based marker system that preferentially targets exonic sequences, which often contain promoters [11]. SRAP analysis has been successfully used in a wide range of purposes, including genetic diversity assessment and germplasm characterization [12—16], map construction [17], gene tagging [18], and comparative genomic analysis [18, 19]. However, reports on the utilization of SRAP in bamboo studies such as genetic diversity assessment are limited. Although China has the largest plant acreage of bamboo in the world, overexploitation and genetic erosion of bamboo species have prompted organizations to establish a bamboo germplasm for conservation purposes. In this context, we collected 13 bamboo species distributed in different regions of China and examined the morphological and molecular relationships among species and genera by using morphological and SRAP analyses.
MATERIALS AND METHODS
Plant materials. Thirteen bamboo species belonging to 5 bamboo genera, Phyllostachys, Pleioblastus Nakai, Shibataea Makino ex Nakai, subgen. Pseu-dosasa, and Bambusa were used in this study (Table 1). These bamboo plants were collected from different regions of China and were grown in the experimental
Table 1. The list of bamboo accessions used in this study
Varieties name Abbr. Genus Cluster Collection site, China
Ma SRAP
Ph. prominens W.Y Xiong PPYX Phyllostachys A I Zhejian
P. gozadakensis Nakai PGNK Pleioblastus Nakai A III Fujian
Ph. heterocycla var. pubescens (Mazel) Ohwi PHVO Phyllostachys B I Fujian
P. amarus var. hangzhouensis S.L. Chen et S. Ychen PASL Pleioblastus Nakai B II Hangzhou
Ph. nigra (Lodd. ex Lindl.)Munro PNMN Phyllostachys B I Hunan
P. intermedius S.Y Chen PLSY Pleioblastus Nakai B III Zhejiang
S. chinensis Nakai SCNK Shibataea Makino ex Nakai B II Jiangsu
P. longinternodius Y.B. Yang PLYY Pleioblastus Nakai A II Guizhou
Ph. rivalis H.R. Zhao et A.T. Liu PRZL Phyllostachys B I Fujian
Ph. bambusoides f. lacrima-deae Keng f. et Wen PBLK Phyllostachys B I Hunan
P. truncatula S.L. Chen et G.Y. Sheng PTSC subgen. Pseudosasa B II Zhejiang
B. multiplex Raeuschel BMRS Bambusa B II Guangdong
B. multiplex cv. Alphonse-Karr BMAK Bambusa B III Sichuan
a Genus is according to Gamble's classification; M is morphological character.
field of the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha, China. Five independent replicates from each species were sampled at random for morphological and DNA analyses. The leaves were separately collected and immediately stored at —80°C until DNA extraction.
Morphological descriptors. Each species was considered as a separate, independent operational taxonom-ic unit (OTU) [20]. Twenty-one key morphological traits, including 15 culm (a type, vine-like stalk, main branch significantly, stalk surface with siliceous, young stalk with white powder, skin with small protuberances, stalk with trenches, striations on culm, sections with spots, curved branches, stalks scattered, spiny branches, square stalk sections, solid stalks, and small transverse veins in the leaves) and 6 culm-sheaths (young stalk with small bristles, green stalk, sheath scars, bristles on auricles, pubescent abaxial and hairs on ligules) were assessed from each of the 13 OTUs. Mean values obtained from 5 independent replicates were used as OTU representative data for each of the qualitative morphological descriptors. The scored qualitative interval data were standardized to construct the dendrogram using the unweighted pair-group
method of arithmetic averages (UPGMA) [21] using NTSYS-pc ver. 2.1 [22].
DNA extraction. DNA was extracted from fresh leaves of each bamboo variety according to the cetylt-rimethyl ammonium bromide (CTAB) method [23] with minor modifications. The DNA was diluted to approximately 10 ng/^L for polymerase chain reaction (PCR) analysis.
SRAP analysis. The SRAP technique consisted of preferential amplification of open reading frames (ORFs) using PCR. For this purpose, combinations of 2 types of primers were employed. The first type of primer (forward) was 17 bp in length and contained a 14-nucleotide GC-rich region and 3 selective bases at its 3' end. This primer preferentially amplifies exonic regions, which are also generally GC-rich. The second type of primer (reverse) was 18 bp in length and contained a 15-nucleotide AT-rich region and 3 selective bases at its 3' end. This primer preferentially amplifies intronic and promoter regions, which are usually AT-rich. Any observed polymorphisms fundamentally reflect variations in the length of these introns, promoters, and spacers, both among individuals and among species [11]. In this study, 86 different combinations were
0.50
0.63
0.75 Coefficient
0.88
PPYX
PGNK
PLYY
PHVO
PASL
SCNK
PNMN
PRZL
PBLK
PLSY
PTSC
BMAK
BMRS
1.00
A
B
Fig. 1. Dendrogram derived from UPGMA cluster analysis based on 22 key morphological characteristics of 13 bamboo species.
employed using 32 forward primers (Me1—Me32) and 26 reverse primers (Em1—Em26) [11, 18]. All the primers were commercially synthesized (Sangon Biological Engineering Technology and Sevice Co. Ltd., Shanghai).
For PCR amplification, each 20-^L PCR reaction mixture consisted of 60 ng of genomic DNA, 30 ng of the primer, 200 ^mol/L of dNTPs, 1.5 mmol/L of MgCl2, 1x Taq buffer, and 1 unit of Taq polymerase (from MBI). For DNA amplification, PCR conditions included the following: the first 5 cycles at 94°C for 1 min, then at 35°C for 1 min, and 72°C for 1 min, for denaturing, annealing, and extension, respectively. The annealing temperature was then raised to 50°C for another 35 cycles [11]. Amplification fragments were separated on a 6% denatured polyacrylamide gel [acrylamide—bisacrylamide (19 : 1) in 0.5x TBE]. The gel was pre-run in 0.5x TBE buffer at 2,000 V constant voltage until the electrical current reached 30 mA prior to sample loading. After loading the samples, the gel was run at 2,500 V constant voltage for approximately 1.5—2 h until the xylene cyanol dye marker had traveled across 2/3 of the gel and the temperature was maintained below 50°C to avoid breakage of the glass gel apparatus. After electrophoresis, the gel was stained with a AgNO3 solution [24].
Data analysis. The amplified products were scored for presence (1) or absence
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