The Open Biotechnology Journal




ISSN: 1874-0707 ― Volume 14, 2020

Transferability of Sorghum Microsatellite Markers to Bamboo and Detection of Polymorphic Markers



Tesfaye Disasa1, *, Tileye Feyissa2, Demissew Sertse3
1 National Agricultural Biotechnology Research Center, P.O. Box 2003, Addis Ababa, Ethiopia
2 Faculty of Life Sciences, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia
3 University of Ottawa, K1Z7P2, 136 Crerar, Ottawa, ON, Canada

Abstract

The use of molecular markers for the characterization and evaluation of plant genetic resources has become a useful approach in plant genetic research. Simple Sequence Repeats (SSRs) are among the markers that are widely used in genetic diversity and parental analysis owing to their co-dominant nature, high reproducibility, abundance in the genome and transferability across species or genera. The development of these markers for a species might be costly and time consuming. Hence, screening existing markers through transferability test from closely related species or family is resource conscious. In this study, the transferability of 90 polymorphic SSR markers of sorghum to bamboo was tested and polymorphic analysis of transferable markers were performed. Nearly 62% of the tested SSRs successfully recorded amplification in at least one bamboo species of which 55% were polymorphic. These polymorphic markers detected a total of 147 alleles at an average rate of 4.7 alleles per marker. The abundant alleles account 20.4% while the common and rare alleles share 39.6 and 40 %, respectively. The result showed a relatively low degree of polymorphic information content (PIC) averaging 0.29. The gene diversity index (He) ranged from 0.21 to 0.49 with a mean of 0.37. The cluster analysis based on the polymorphic markers surfaced most of the species in accordance with their geographic origin. The complementarity of the weighted neighbour joining tree and coordinate analysis implies the representative nature of the transferred markers for the diversity analysis of bamboo species.

Keywords: Bamboo, genetic diversity, PCR analysis, polymorphic information content, sorghum, SSR markers, transferability.


Article Information


Identifiers and Pagination:

Year: 2016
Volume: 10
First Page: 223
Last Page: 233
Publisher Id: TOBIOTJ-10-223
DOI: 10.2174/18740707016100100223

Article History:

Received Date: 13/11/2015
Revision Received Date: 4/1/2016
Acceptance Date: 22/1/2016
Electronic publication date: 13/05/2016
Collection year: 2016

© Disasa; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution-Non-Commercial 4.0 International Public License (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/legalcode), which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.


* Address correspondence to this author at the National Agricultural Biotechnology Research Center, P.O. Box 2003, Addis Ababa, Ethiopia; Tel: +251 911118068; Fax: +251 112370377; E-mail: tesfayedisasa@yahoo.com





INTRODUCTION

Bamboos are members of the sub-family Bambusoideae within the grass family Poaceae [1Soderstrom TR, Ellis RP. The position of bamboo genera and allies in a system of grass classification Grass Systematics and Evolution. Washington, DC: Smithsonian Institution Press 1987; pp. 225-38.] in which most important cereal crops such as rice, wheat, sorghum, maize and barley are also grouped. Bambusoideae is a large sub-family containing more than 70 genera with over 1450 species [2Hunter IR. Bamboo resources, uses and trade: the future? J Bamboo Rattan 2003; 2(4): 319-29.
[http://dx.doi.org/10.1163/156915903322700368]
]. It is a fast growing wood grass distributed all over the world, but major contributors of bamboo resource are Asia (particularly China, India Japan, Myanmar and Malaysia) and South America mainly Brazil, Venezuela and Colombia [3Embaye K. The indigenous bamboo forests of Ethiopia: An Overview. Ambio 2000; 29: 518-21.
[http://dx.doi.org/10.1579/0044-7447-29.8.518]
]. Ethiopia ranks first in Africa which comprises 67 % of the total bamboo area coverage of the continent [4Das M, Bhattacharya S, Pal A. Generation and characterization of SCARs by cloning and sequencing of RAPD products: a strategy for species-specific marker development in bamboo. Ann Bot (Lond) 2005; 95(5): 835-41.
[http://dx.doi.org/10.1093/aob/mci088] [PMID: 15731116]
].

Bamboo is an important multipurpose genetic resource in the world for its wide range of economic values. It is the source of building materials, high quality furniture, food, fiber, fodder, pulp, paper, board and charcoal [5Das M, Bhattacharya S, Singh P, Filgueiras TS, Pal A. Bamboo taxonomy and diversity in the era of molecular markers. Adv Bot Res 2008; 47: 225-68.
[http://dx.doi.org/10.1016/S0065-2296(08)00005-0]
-8Nayak S, Rout GR. Isolation and characterization of microsatellites in Bambusa arundinacea and cross species amplification in other bamboos. Plant Breed 2005; 124: 599-602.
[http://dx.doi.org/10.1111/j.1439-0523.2005.01102.x]
]. It also serves as ornamental plant. The plant also plays a significant role in ecological applications because of unlimited environmental values [2Hunter IR. Bamboo resources, uses and trade: the future? J Bamboo Rattan 2003; 2(4): 319-29.
[http://dx.doi.org/10.1163/156915903322700368]
]. Due to its fast growth nature, it can be harvested within short period of time without depletion and deterioration of the soil. It can grow on marginal land which is not suitable for agriculture or forestry. In addition, the plant serves as source of feed for many wild animals.

Despite its huge importance of the plant, it is less studied and its genetic diversity is little understood. Besides, very wide area coverage in western and south western Ethiopia currently are severely affected as a result of mass flowering and seed setting of the plant [9Sertse D, Disasa T, Bekele K, et al. Mass flowering and death of bamboo: a potential threat to biodiversity and livelihoods in Ethiopia. J Biod Env Sci 2011; 1(5): 16-25.]. It demands extensive research to exploit the effective methods for characterization and conservation of this important genetic resource. Morphological traits are the most commonly used approach in bamboo classification [3Embaye K. The indigenous bamboo forests of Ethiopia: An Overview. Ambio 2000; 29: 518-21.
[http://dx.doi.org/10.1579/0044-7447-29.8.518]
]. Although morphological traits remain to be major characterization tools, the recent advance in molecular genetics has enabled in refining classification and within species diversity analysis at higher resolution.

Various marker systems such as random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), diversity arrays technology (DArT) and single nucleotide polymorphism (SNP) have been used in the study of various plant genetic resources. Owing to the technical simplicity in their application, recently, SSRs have become major tools in population genetics, genetic mapping and other plant genomic studies [10Goldstein D, Schlo¨terer C. Microsatellite: Evolution and applications. Oxford: Oxford University Press 1999; p. 368.]. These markers are highly reproducible, which is highly important in genetic analysis. They are highly polymorphic and produce very high allelic variations even among very closely related varieties. The co-dominance nature of the marker also helps to analyze the segregating populations or paternity testing. The SSR markers are abundant and well distributed throughout the nuclear genome of the species [11Gaitan-Solis E, Duque MC, Edwards KJ, Tohme J. Microsatellite repeats in common bean (Phaseolus vulgaris): isolation, characterization, and cross-species amplification in Phaseolus ssp. Crop Sci 2002; 42: 2128-36., 12Saha MC, Mian MA, Eujayl I, Zwonitzer JC, Wang L, May GD. Tall fescue EST-SSR markers with transferability across several grass species. Theor Appl Genet 2004; 109(4): 783-91.
[http://dx.doi.org/10.1007/s00122-004-1681-1] [PMID: 15205737]
]. However, the development of SSR markers requires relatively high cost and time consuming [13Squirrell J, Hollingsworth PM, Woodhead M, et al. How much effort is required to isolate nuclear microsatellites from plants? Mol Ecol 2003; 12(6): 1339-48.
[http://dx.doi.org/10.1046/j.1365-294X.2003.01825.x] [PMID: 12755865]
, 14Zane L, Bargelloni L, Patarnello T. Strategies for microsatellite isolation: a review. Mol Ecol 2002; 11(1): 1-16.
[http://dx.doi.org/10.1046/j.0962-1083.2001.01418.x] [PMID: 11903900]
]. On account of their transferability across related families, SSR developed for one species can be used for other species within same family.

Microsatellites are usually transferable across grass family due to the evolutionary relationships among species belong to the same family. SSR markers particularly expressed sequence tag (EST)-derived SSRs have shown to possess a higher potential for inter-specific transferability than genomic SSRs [15Sharma V, Bhardwaj P, Kumar R, Sharma RK, Sood A, Ahuja PS. Identification and cross-species amplification of EST derived SSR markers in different bamboo species. Conserv Genet 2009; 10: 721-4.
[http://dx.doi.org/10.1007/s10592-008-9630-1]
]. Several studies have been conducted on the transferability of SSR markers across species or genera of several cereal crops which belong to grass family such as rice [16Zhao X, Kochert G. Phylogenetic distribution and genetic mapping of a (GGC)n microsatellite from rice (Oryza sativa L.). Plant Mol Biol 1993; 21(4): 607-14.
[http://dx.doi.org/10.1007/BF00014544] [PMID: 8448360]
], wheat [17Röder MS, Plaschke J, König SU, et al. Abundance, variability and chromosomal location of microsatellites in wheat. Mol Gen Genet 1995; 246(3): 327-33.
[http://dx.doi.org/10.1007/BF00288605] [PMID: 7854317]
], wheat, rye and triticale [18Kuleung C, Baenziger PS, Dweikat I. Transferability of SSR markers among wheat, rye, and triticale. Theor Appl Genet 2004; 108(6): 1147-50.
[http://dx.doi.org/10.1007/s00122-003-1532-5] [PMID: 15067402]
], barley [19Thiel T, Michalek W, Varshney RK, Graner A. Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor Appl Genet 2003; 106(3): 411-22.
[PMID: 12589540]
], sugar cane [20Cordeiro GM, Casu R, McIntyre CL, Manners JM, Henry RJ. Microsatellite markers from sugarcane (Saccharum spp.) ESTs cross transferable to erianthus and sorghum. Plant Sci 2001; 160(6): 1115-23.
[http://dx.doi.org/10.1016/S0168-9452(01)00365-X] [PMID: 11337068]
, 21Banumathi G, Krishnasamy V, Maheswaran M, Samiyappan R, Govindaraj P, Kumaravadivel N. Genetic diversity analysis of sugarcane (Saccharum sp.) clones using simple sequence repeat markers of sugarcane and rice. Electro J Plant Breed 2010; 1: 517-26.], sorghum [22Brown SM, Hopkins MS, Mitchell SE, et al. Multiple methods for the identification of polymorphic simple sequence repeats (SSRs) in sorghum [Sorghum bicolor (L.) Moench]. Theor Appl Genet 1996; 93(1-2): 190-8.
[http://dx.doi.org/10.1007/BF00225745] [PMID: 24162217]
, 23Savadi SB, Fakrudin B, Nadaf HL, Gowda MV. Transferability of sorghum genic microsatellite markers to peanut. American J Plant Sci 2012; 3: 1169-80.
[http://dx.doi.org/10.4236/ajps.2012.39142]
] major cereal to minor grass [24Wang LM, Barkleyl NA, Yu JK, et al. Transfer of simple sequence repeat (SSR) markers from major cereal crops to minor grass species for germplasm characterization and evaluation. Plant Genet Res 2005; 3: 45-57.
[http://dx.doi.org/10.1079/PGR200461]
] and pearl millet [25Yadav OP, Mitchell SE, Fulton TM, Kresovich S. Transferring molecular markers from sorghum, rice and other cereals to pearl millet and identifying polymorphic markers. J SAT Agri Res 2008; 6: 1-4.]. There are also reports about transferability of SSRs from cereals such as rice and sugar cane to bamboo species [26Chen SY, Lin YT, Lin CW, Chen WY, Yang CH, Ku HM. Transferability of rice SSR markers to bamboo. Euphytica 2010; 175: 23-33.
[http://dx.doi.org/10.1007/s10681-010-0159-2]
, 27Sharma RK, Gupta P, Sharma V, Sood A, Mohapatra T, Ahuja PS. Evaluation of rice and sugarcane SSR markers for phylogenetic and genetic diversity analyses in bamboo. Genome 2008; 51(2): 91-103.
[http://dx.doi.org/10.1139/G07-101] [PMID: 18356943]
]. Sharma et al. [15Sharma V, Bhardwaj P, Kumar R, Sharma RK, Sood A, Ahuja PS. Identification and cross-species amplification of EST derived SSR markers in different bamboo species. Conserv Genet 2009; 10: 721-4.
[http://dx.doi.org/10.1007/s10592-008-9630-1]
] also conducted an experiment on identification and amplification of EST-SSR markers in different bamboo species. Sorghum is one of the cereals crops with already developed large number of SSRs markers and it is genetically closer to bamboos than other cereals such as rice. However, there has been no report on transferability of sorghum SSR markers to bamboo species. The objective of this study was to perform the transferability test of SSR markers from sorghum to bamboo species and to employ the transferable markers in the genetic diversity analysis of selected bamboo species.

MATERIALS AND METHODS

Plant Materials and DNA Isolation

Fresh leaf tissues from seven exotic and two indigenous Ethiopian bamboos representing both temperate and tropical species were collected (Table 1). Five leaves from each of the species were bulked and lyophilized prior to DNA extraction. Genomic DNA was extracted using Wizard® Genomic DNA Purification Kit (Promega, USA). Genomic DNA samples from sorghum were also included as control. The DNA concentration was quantified by spectrophotometer (Thermo Scientific, USA) and the quality was checked using 0.8% (w/v) agarose gel stained with GelRed® (Biotium, USA). The samples were dissolved with pure sterilized water and the final volume was diluted to 10 ng/µl. Both genomic and PCR product bands were viewed using gel image system (Uvitec, UK).

Table 1

Bamboo species that are used as test genotypes.




HARC (Holetta Agricultural Research Center) is one of the oldest and biggest Agricultural Research Centers in Ethiopia located 38°30′E, 9°4′N with an altitude of 2400 m.a.s.l. receiving annual average rainfall of 1100 mm with minimum and maximum temperature of 6 & 22 ºC, respectively.

Selection of Sorghum SSR Markers and PCR Analysis

A pair of 90 SSR polymorphic sorghum primers [28Menz MA, Klein RR, Mullet JE, Obert JA, Unruh NC, Klein PE. A high-density genetic map of Sorghum bicolor (L.) Moench based on 2926 AFLP, RFLP and SSR markers. Plant Mol Biol 2002; 48(5-6): 483-99.
[http://dx.doi.org/10.1023/A:1014831302392] [PMID: 11999830]
-31Wu YQ, Huang Y. An SSR genetic map of Sorghum bicolor (L.) Moench and its comparison to a published genetic map. Genome 2007; 50(1): 84-9.
[http://dx.doi.org/10.1139/g06-133] [PMID: 17546074]
] that are evenly distributed across the whole sorghum nuclear genome were selected to test the transferability of the marker across the selected bamboo species (Table 2). PCR amplification was performed in a 10 µl reaction volume comprising of 1 x PCR buffer (20 mM Tris-HCl, pH 7.6; 100 mM KCl; 0.1 mM EDTA; 1 mM DTT; 0.5% (w/v) Triton X-100; 50% (v/v) glycerol), 2 mM MgCl2, 0.16 mM dNTPs, 0.16 µM fluorescent labeled M13-forward primer, 0.04 µM forward primer, 0.2 µM reverse primer, 0.2 units of Taq DNA polymerase (SibEnzyme Ltd, Russia) and 30 ng of template DNA. All forward primers contained an M13-tag (5’- CACGACGTTGTAAAACGAC - 3’) on the 5’ end that was fluorescently labeled to allow detection of amplification products. The Forward primers were labelled with FAM, PET, NED or VIC (Applied Biosystems). PCR reactions were carried out in a GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems, USA) programmed for initial denaturation at 94°C for 15 min, followed by second denaturation at 94°C for 30 sec, annealing temperature ranging from 50°C to 55°C for 1 min, extension at 72°C for 2 min and final elongation at 72°C for 20 min.

Fig. (1)

Informative sorghum microsatellites showing high rate of transferability across the tested bamboo species. Amplicons were generated from genomic DNA of Arundinaria alpine by PCR at 55 °C annealing temperature. The fragment was separated on 2% (w/v) agarose gel along with 1 kb ladder. Xiabtp424 didn’t show any band and hence used as a negative control.



Successful amplification was confirmed by running 2.0 µl of the PCR products on a 2% (w/v) agarose gel stained with GelRed® (Biotium) and visualized using gel image system (UVtech, UK). Annealing temperature for the selected bamboo species were optimized in order to obtain clear bands.

Clear and strong bands were observed at annealing temperature 55°C (Fig. 1) while the optimum annealing temperature for most sorghum polymerase chain reaction is 50°C. The success of PCR amplification is largely dependent on the annealing temperature of the PCR program. Optimization of the program for the selected experiment is a crucial step during the analysis. The optimized annealing temperature (55°C) can be applied to amplify the DNA of various bamboo species and other related polyploidy species.

Table 2

List of selected sorghum SSR markers [28Menz MA, Klein RR, Mullet JE, Obert JA, Unruh NC, Klein PE. A high-density genetic map of Sorghum bicolor (L.) Moench based on 2926 AFLP, RFLP and SSR markers. Plant Mol Biol 2002; 48(5-6): 483-99.
[http://dx.doi.org/10.1023/A:1014831302392] [PMID: 11999830]
-31Wu YQ, Huang Y. An SSR genetic map of Sorghum bicolor (L.) Moench and its comparison to a published genetic map. Genome 2007; 50(1): 84-9.
[http://dx.doi.org/10.1139/g06-133] [PMID: 17546074]
] tested for their transferability to bamboo.




In order to perform the genetic diversity and relatedness among the target species, the successfully transferable markers were genotyped using capillary electrophoresis. Depending on the nature of fluorescent label and strength of the amplification bands used, a volume ranging from 2.5 µl to 3.5 µl of four different amplification products were co-loaded along with the internal size standard, GeneScan™ -500 LIZ® (Applied Biosystems) and Hi-Di™ Formamide (Applied Biosystems, USA). The fragments were denatured at 95°C for five minutes using GeneAmp® PCR System 9700 thermal cycler. The fragments were separated by capillary electrophoresis using an ABI Prism® 3730 Genetic analyzer (Applied Biosystems) in order to determine the exact allele size of each fragment.

Data Analysis

PCR fragment size was manually scored using GeneMapper v4.0 software. The allele size in terms of base pairs was compared to the internal size standard, GeneScan™ -500 LIZ® (Applied Biosystems, USA). Percentage of transferability was computed by dividing the number of the SSR markers amplified in non-donor (bamboo) species to the total number of tested SSRs. Fragments in base pair were converted to binary data because of the complex amplification pattern in bamboo genome [27Sharma RK, Gupta P, Sharma V, Sood A, Mohapatra T, Ahuja PS. Evaluation of rice and sugarcane SSR markers for phylogenetic and genetic diversity analyses in bamboo. Genome 2008; 51(2): 91-103.
[http://dx.doi.org/10.1139/G07-101] [PMID: 18356943]
]. The level of polymorphism of the entire markers across the selected bamboo species was computed. Only polymorphic markers were selected to analyze the genetic diversity and relatedness among the target species. The proportions of polymorphic loci were calculated as:

Where,

P = proportion of polymorphic loci

npj = number of polymorphic loci

ntotal = total number of loci

Power Marker v.3.25 [32Liu K, Muse SV. PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics 2005; 21(9): 2128-9.
[http://dx.doi.org/10.1093/bioinformatics/bti282] [PMID: 15705655]
] was used to compute PIC and allele frequencies. Rare, common and abundant were manually computed in MS Excel (Microsoft Inc., Seattle, USA). Polymorphism information content (PIC) was calculated using the method of Botstein et al. [33Botstein D, White RL, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 1980; 32(3): 314-31.
[PMID: 6247908]
].

Where, pi and pj are the frequencies of alleles i and j, respectively

Markers with a PIC value of more than 0.5 were considered highly informative, between 0.25 and 0.5 as informative and less than 0.25 as less informative.

Pair-wise genetic differentiation between the selected bamboo species, a genetic dissimilarity matrix was calculated using simple matching with DARwin v5 software (available at http://darwin.cirad.fr/darwin/Home.php). Individual relations were analyzed with a tree construction based on Neighbor Joining (NJ) method, as implemented in DARwin v5.

RESULTS AND DISCUSSION

The availability of large number of sorghum SSR markers as a community resource and the consideration of sorghum as a model crop to study other grass family created a scope of opportunity to exercise similar methodology in non-model plants like bamboo. The current advancement in genomic technology will also help to set the best conservation strategies particularly for the endangered plant genetic resource as well as economically important plants like bamboo. To achieve this magnificent plan of genetic resource conservation, it is imperative to clearly characterize the core collection prior to conservation practices.

Among 90 tested sorghum SSR markers, 62.2% of them scored successful amplification in a bamboo representative species, Arundinaria alpine (Table 3). However, transferability rate differed among bamboo species. The lowest transferability rate that accounted only 40% was observed in Gigantochloa atter. Markers with high rate of transferability include Xtxp023, Xtxp205, Xtxp283 and Xcup16 where as Xiabtp346 and Xisep0327 showed reduced transferability rate. Markers such as Xiabtp424, Xise0443 and Xisep0648 did not score any amplification in either of the bamboo species. The transferability rate obtained in this study is higher than the one reported for various grass family species including bamboo using [18Kuleung C, Baenziger PS, Dweikat I. Transferability of SSR markers among wheat, rye, and triticale. Theor Appl Genet 2004; 108(6): 1147-50.
[http://dx.doi.org/10.1007/s00122-003-1532-5] [PMID: 15067402]
, 26Chen SY, Lin YT, Lin CW, Chen WY, Yang CH, Ku HM. Transferability of rice SSR markers to bamboo. Euphytica 2010; 175: 23-33.
[http://dx.doi.org/10.1007/s10681-010-0159-2]
, 27Sharma RK, Gupta P, Sharma V, Sood A, Mohapatra T, Ahuja PS. Evaluation of rice and sugarcane SSR markers for phylogenetic and genetic diversity analyses in bamboo. Genome 2008; 51(2): 91-103.
[http://dx.doi.org/10.1139/G07-101] [PMID: 18356943]
]. However, the rate is slightly lower than the rice SSR transferability to bamboo that was reported to be 68% [26Chen SY, Lin YT, Lin CW, Chen WY, Yang CH, Ku HM. Transferability of rice SSR markers to bamboo. Euphytica 2010; 175: 23-33.
[http://dx.doi.org/10.1007/s10681-010-0159-2]
]. It is possible to improve the transferability rate of markers by using markers that were developed from expressed sequences [18Kuleung C, Baenziger PS, Dweikat I. Transferability of SSR markers among wheat, rye, and triticale. Theor Appl Genet 2004; 108(6): 1147-50.
[http://dx.doi.org/10.1007/s00122-003-1532-5] [PMID: 15067402]
].

The scored fragment size ranged between 104 bp and 486 bp. Most of these fragments were amplified higher than the size of the donor fragment, which is in agreement with the previous study implying that the amplified fragments from the selected species have different allele sizes than those of the donor species [26Chen SY, Lin YT, Lin CW, Chen WY, Yang CH, Ku HM. Transferability of rice SSR markers to bamboo. Euphytica 2010; 175: 23-33.
[http://dx.doi.org/10.1007/s10681-010-0159-2]
, 24Wang LM, Barkleyl NA, Yu JK, et al. Transfer of simple sequence repeat (SSR) markers from major cereal crops to minor grass species for germplasm characterization and evaluation. Plant Genet Res 2005; 3: 45-57.
[http://dx.doi.org/10.1079/PGR200461]
, 27Sharma RK, Gupta P, Sharma V, Sood A, Mohapatra T, Ahuja PS. Evaluation of rice and sugarcane SSR markers for phylogenetic and genetic diversity analyses in bamboo. Genome 2008; 51(2): 91-103.
[http://dx.doi.org/10.1139/G07-101] [PMID: 18356943]
].

Out of the 56 SSR markers that were found to be transferable, 31 (55%) of the showed polymorphism. A total of 147 SSR marker alleles were recovered, at an average rate of 4.5 alleles per marker. The average number of fragments per marker is less than the previous transferability test conducted using rice and sugar cane SSRs markers to study bamboo genetic diversity [27Sharma RK, Gupta P, Sharma V, Sood A, Mohapatra T, Ahuja PS. Evaluation of rice and sugarcane SSR markers for phylogenetic and genetic diversity analyses in bamboo. Genome 2008; 51(2): 91-103.
[http://dx.doi.org/10.1139/G07-101] [PMID: 18356943]
]. The number of alleles per marker ranged from 2 to 12 with the highest number of alleles obtained from Xisep0138 followed by Xcup24 and Xiabtp240 (Table 4). The presence of these multiple amplification products could be due to duplication, high level of polyploidy and genetic variability in bamboo species [12Saha MC, Mian MA, Eujayl I, Zwonitzer JC, Wang L, May GD. Tall fescue EST-SSR markers with transferability across several grass species. Theor Appl Genet 2004; 109(4): 783-91.
[http://dx.doi.org/10.1007/s00122-004-1681-1] [PMID: 15205737]
, 27Sharma RK, Gupta P, Sharma V, Sood A, Mohapatra T, Ahuja PS. Evaluation of rice and sugarcane SSR markers for phylogenetic and genetic diversity analyses in bamboo. Genome 2008; 51(2): 91-103.
[http://dx.doi.org/10.1139/G07-101] [PMID: 18356943]
, 34Yu JK, Dake TM, Singh S, et al. Development and mapping of EST-derived simple sequence repeat markers for hexaploid wheat. Genome 2004; 47(5): 805-18.
[http://dx.doi.org/10.1139/g04-057] [PMID: 15499395]
]. Among the polymorphic markers (n=31), 15 of them showed a clear band and highly transferable to all the bamboo species tested in this experiment (Fig. 1). These markers are found to be very effective and useful resources to utilize them for phylogenetic and genetic diversity studies of the different bamboo species. In contrary, very few markers were amplified only in some specific bamboo species ( Supplementary Table S1).

A total of 59 alleles (40 %) were detected as rare alleles with an average of two alleles per marker. The highest number of rare alleles was obtained for marker Xcup24. Similarly, 58 alleles (39.5%) scored common allele frequency considered as common alleles with an average of two alleles per marker. Xisep0327 showed the highest number of common allele. The remaining 30 alleles (20.5%) were found to be abundant allele with an average number of one allele per marker. The higher frequencies of rare and common alleles are very important resources for future bamboo characterization and maintenance program. Even though, the significance of the rare alleles cannot be speculated from the current results, their abundance in the selected bamboo species suggests that this diverse set of genetic resource has not been extensively exploited.

The level of polymorphism detected across species or genera mainly depends on the genetic divergence of species tested and primers used [24Wang LM, Barkleyl NA, Yu JK, et al. Transfer of simple sequence repeat (SSR) markers from major cereal crops to minor grass species for germplasm characterization and evaluation. Plant Genet Res 2005; 3: 45-57.
[http://dx.doi.org/10.1079/PGR200461]
]. The PIC values for the SSR loci ranged from 0.19 to 0.37 with an average of 0.29 (Table 4). Five markers such as Xtxp205, Xtxp057, Xiabtp310, Xtxp040 and Xiabtp340 showed the highest PIC value of 0.37 whereas the lowest PIC value of 0.19 was recorded for marker Xcup24. The gene diversity index (expected heterozygosity, He) ranged from 0.21 to 0.49 with a mean of 0.37. Marker Xcup24 presented the lowest gene diversity as well as PIC value. Though the computed average PIC value in this experiment was relatively smaller, it would be sufficient to utilize in characterization of bamboo collection. The wide variation in ploidy level among the different bamboo species has created the generation of multiple alleles derived from a single locus. This has implication that a small number of markers can discriminate the different bamboo species and a useful aspect towards the conservation of the genetic resources with rare allele.

Table 3

Rate of sorghum SSR transferability across the selected Bamboo species.




The highest genetic distance (0.90) was observed between Guadua amplexita, Gigantochloa apus and Guadua amplexita, Gigantochloa atter while lowest genetic distance (0.32) was recorded between Gigantochloa apus and Gigantochloa sumatra (Table 5). Relatively higher genetic distance (0.77) was obtained between Ethiopian lowland (Oxytenanthera abyssinica) and highland bamboo (Arundinaria alpine). The distinctiveness of these species requires proper conservation techniques in order to maintain the genetic resources.

Genetic analysis based on the polymorphic markers successfully grouped the different species in accordance to the geographic origin of the species and their genera (Fig. 2). This study revealed that Ethiopian bamboos which exist in different genera are highly differentiated. The dandrogram generated from Neighbor-Joining (NJ) tree analysis was in congruent with the result obtained from the principle coordinate analysis with few exceptions (Figs. 2, 3). The similarity in the clustering of these bamboo species using these two approaches further validates the representative nature of the transferred markers for the diversity analysis of bamboo species. The current study will serve as a foundation to study bamboo genetic resources using the transferable markers and hence exhaustive collection and characterization of both highland and lowland bamboo from the entire growing regions will be necessary in the future.

Fig. (2)

Phylogenetic groups of selected bamboo species based on selected 31 polymorphic SSR loci.



Fig. (3)

Biplot of the axis 1 and 2 of the principle coordinate analysis based on the dissimilarity of 31 SSR markers.



Table 4

Basic statistics of 31 sorghum microsatellites markers that are found to be polymorphic and transferable to selected bamboo species.




CONCLUSION

The transferability of SSR markers from related families is an alternative approach to bypass the cost developing species specific markers as well as the complexity of the work. The current screened sorghum SSR markers for the purpose of bamboo research will provide a scope of opportunity to researchers for the proper characterization and conservation of bamboo genetic resources. The adequate rate of polymorphism in the markers helps to characterize large number of materials with limited SSR markers. This work will be a foundation particularly in Ethiopia which contributes 67% of the African bamboo area coverage to develop strategies for characterization, and conservation of bamboo genetic resource using molecular approaches. The higher level of rare alleles across both exotic and Ethiopian bamboo species plays an important role in further study of these alleles in detail. Such markers will also guide the implementation of various conservation decisions that will be necessary to ensure that these rare alleles are not lost.

Table 5

Pair-wise genetic distance between the selected bamboo species.




SUPPLEMENTARY MATERIAL

Supplementary material is available on the publishers Website along with the published article.

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CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

The expenses of this research were fully covered by the Swedish International Development Agency (SIDA) through Bio-Innovate project “Delivering new sorghum and millets innovations for food security and improving livelihoods in Eastern Africa”- project No. 01/2010. We also gratefully acknowledge the forestry research team of Holetta Agricultural Research Center of the Ethiopian Institute of Agricultural Research for allowing us their bamboo collection garden.

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