Isolation and characterization of microsatellite markers for Acacia tortilis (Forsk.) Hayne Stephen Fredrick Omondi, Joseph Machua, John Gicheru & So Hanaoka
Conservation Genetics Resources ISSN 1877-7252 Conservation Genet Resour DOI 10.1007/s12686-014-0413-3
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Author's personal copy Conservation Genet Resour DOI 10.1007/s12686-014-0413-3
MICROSATELLITE LETTERS
Isolation and characterization of microsatellite markers for Acacia tortilis (Forsk.) Hayne Stephen Fredrick Omondi • Joseph Machua John Gicheru • So Hanaoka
•
Received: 12 December 2014 / Accepted: 23 December 2014 Ó Springer Science+Business Media Dordrecht 2015
Abstract Acacia tortilis is a drought tolerant multipurpose dryland tree species widely distributed in Africa and Asia. Its overexploitation has caused its population decrease. A set of 11 polymorphic nuclear microsatellite loci were isolated and characterized for A. tortilis using samples from three natural populations from Kenya. All the loci were polymorphic and on average, 14 alleles per locus were detected ranging from 12 to 16. Expected and observed heterozygosity ranged from 0.681 to 0.856 and 0.656–0.85 respectively. These microsatellite markers will provide valuable genetic data for studies and sustainable management strategy for A. tortilis. Keywords Acacia tortilis Multipurpose tree Genetic diversity Microsatellite Population Conservation Acacia tortilis (Forsk.) Hayne is one of the approximately 135 African acacia species. The species is drought tolerant and is widely distributed throughout Africa and Middle East. Its habitat ranges from arid to semi-arid lands characterized by dry bushlands and grasslands. Morphologically it is variable ranging from branchy small tree in the grasslands to a large giant tree along the riverine. The species is majorly used Electronic supplementary material The online version of this article (doi:10.1007/s12686-014-0413-3) contains supplementary material, which is available to authorized users. S. F. Omondi (&) J. Machua J. Gicheru Kenya Forestry Research Institute, P.O Box 20412-00200, Nairobi, Kenya e-mail:
[email protected] S. Hanaoka Forest Tree Breeding Center, Forestry and Forest Products Research Institute, Hitachi, Ibaraki 319-1301, Japan
for charcoal production, source of livestock fodder and soil fertility improvement. Despite the ecological and economic potential of the species, it is overexploited and little is known about its current genetic status. Furthermore only 12 sets of nuclear microsatellite molecular markers have been developed for it (Winters et al. 2013). There is need to develop more informative molecular makers to enable studies of the species population genetics for sustainable management. This paper reports the development and characterization of a set of 11 polymorphic microsatellite loci for A. tortilis. Two plant leaf samples were used as the source of DNA for genomic library construction. Genomic DNA was isolated from silica dried leaf samples using DNeasy Plant Mini Kit (QIAGEN). Microsatellite loci were isolated using enrichment method described in Hamilton et al. (1999) with some modifications as described in Hanaoka et al. (2012). Enriched DNA fragments were recovered as in Hanaoka et al. (2012) and amplified PCR products purified using QIAquick PCR Purification Kit (QIAGEN) and cloned using PT7 Blue T-vector (Nobagen). A total of 192 colonies were selected and sequenced using BigDye Terminator Cycle Sequencing Kit Ver. 3.1 with universal T7 or U19 primers using ABI 3130xl sequencer (Applied Biosystems). Out of 192 clones, 144 containing SSR motif with adequate flanking regions for primer development were selected. Primer pairs were designed using Primer3Plus (Untergasse et al. 2007; http://www.bioinformatics.nl/ cgi-bin/primer3plus/primer3plus.cgi). After testing, 11 microsatellite markers (Table 1) were kept and used to characterize 90 samples of A. tortilis from three natural populations (Voi, Bura and Isiolo) from Kenya. Polymerase chain reaction (PCR) was performed in a final volume of 10 ll containing 29 Multiplex PCR Master Mix (QIAGEN), 0.15 lM VIC-labeled M13(-21) primer
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kfat004
kfat033
kfat034
kfat037
kfat051
kfat069
kfat083
kfat086
kfat103
kfat119
kfat142
KP256346
KP256347
KP256348
KP256349
KP256350
KP256351
KP256352
KP256353
KP256354
KP256355
KP256356
(CT)8 (CA)9
F: CATGGATGGATCTAGCAGCCa
(AC)8
F: AGGCTTTTGGACCGAAGAAGa
R: GAGCGTGACTGGCTATTTGA
(AC)22
F: CGTAACAAGGGAGGCTTGTAa R: ATTGTCGGTCTTATGCCAGT
R: TTCAGCTGCTTCGTTTCTGT
F: ATCGTATCGATAAGCACCGCa
R: GGGTTGTGTGTTTGTGTGTG (AG)10
(AC)13
F: ACGAGAAGAGTATGCAAGCCa
R: TTACGGCGCCTAACTATTCG
(CA)14
(TG)10
(CT)16
F: GCTCCATTGCTCCATGGTTAa
R: AGGAAGAAAATGGGTTTTGG
F: AACTCGATTTTCCAATGCAGa
R: TTCTGGGATTCGATTCTTTG
F: TTCCCAGTCAGAGTTCAACAa
R: ACTGGTTCACAAATGCTCGT
F: AAGTCTTTTTCACCCCACCCa (TG)10
(AC)9
F: CACACACGAAGGCTTCTTCTa
R: TCTGTCAGGAACGAGCTACA
(CA)11
F: CAGTCACCACCGAACAATGAa R: TCTGAGCGGACAATGTGATT
R: ACGCCAATATTGCACTCCAT
Motif repeat
Primer sequences (50 –30 )
243–265
236–282
233–277
144–186
161–207
241–277
240–274
249–271
230–252
204–244
232–280
Allele size range (bp)
13
15
14
15
14
15
15
11
12
16
15
Na
0.743
0.707
0.889
0.738
0.826
0.842
0.776
0.786
0.759
0.690
0.793
HO Voi (ns)
0.751 ns
0.643 ns
0.913 ns
0.712*
0.679*
0.743 ns
0.715 ns
0.767 ns
0.733 ns
0.786 ns
0.724 ns
HO Isiolo
0.793
0.891
0.767
0.867
0.746
0.893
0.782
0.721
0.775
0.787
0.835
HO Bura (ns)
0.832
0.756
0.905
0.795
0.906
0.861
0.815
0.814
0.847
0.897
0.791
HE Voi
0.744
0.729
0.938
0.814
0.855
0.769
0.762
0.774
0.772
0.896
0.858
HE Isiolo
0.744
0.875
0.861
0.833
0.863
0.834
0.799
0.817
0.791
0.778
0.757
HE Bura
50 M13 tail: TGTAAAACGACGGCCAGT, F forward sequence, R reverse sequence, Na number of observed alleles per locus, HO heterozygosity observed with P-values for the Hardy– Weinberg equilibrium test and significance threshold adjusted using the Bonferroni correction: P [ 0.05 corresponding to ns- not significant, *P \ 0.05, HE heterozygosity expected
a
Locus name
Accession no. NCBI GenBank
Table 1 Descriptive statistics over all loci for the three natural populations of Acacia tortilis
Author's personal copy Conservation Genet Resour
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(Applied Biosystems), 0.01 lM M13-tailed forward primer and 0.15 lM reverse primer, and 10 ng of template DNA. The following PCR program was used; initial denaturation at 95 °C for 15 min, followed by 30 cycles at 94 °C for 30 s, 58 °C for 90 s and 72 °C for 90 s, and a single final cycle at 60 °C for 30 min using Verity 96 well thermal cycler (Applied Biosystems). Amplified fragments were analysed against an internal standard (Liz 600 size standard) on an ABI 3500 sequencer (Applied Biosystems). The alleles were scored using GeneMapper 5.0 (Applied Biosystems). Genetic parameters were determined using FSTAT (Goudet 2001). Hardy–Weinberg expectations (HWE) and linkage disequilibrium (LD) were also tested. The number of alleles per locus ranged from 11 (kfat037) to 16 (kfat033) (Table 1). Heterozygosity ranged from 0.756 (kfat119) to 0.906 (kfat083) in the Voi population, from 0.729 (kfat119) to 0.938 (kfat103) in the Isiolo population and from 0.744 (kfat142) to 0.875 (kfat119) in the Bura population (Table 1). Total paternity exclusion probability (Pe) over all loci was 0.999. Two pairs of loci (kfat103–kfat004 and kfat083–kfat086) showed significant LD at 5 % level after Bonferroni correction. Deviation from HWE was only detected for two loci in Isiolo population (Table 1). Microsatellite loci selected in this study constitute an efficient tool to investigate genetic diversity and structure of A. tortilis populations. They will be used to assess the mating systems, gene flow, parentage and population dynamics of the species. Results will be useful in the
implementation of conservation and management strategies of the species. Acknowledgments This study was funded and conducted by collaboration between Kenya Forestry Research Institute (KEFRI) and Forest Tree Breeding Centre (FTBC) and Forest and Forest Products Institute (FPRRI) of Japan under KEFRI/JICA ‘‘Project on development of drought tolerant trees for adaptation to climate change in drylands of Kenya’’. We are grateful to KEFRI Biotechnology and Forest FTBC laboratory staff for their support during the study.
References Goudet J (2001) FSTAT, a program to estimate and test gene diversities and fixation indices (version 2.9.3). Updated from Goudet (1995). Available from http://www.unilch/izea/soft wares/fstat.html Hamilton MB, Pincus EL, Di Fiore A, Fleischer RC (1999) Universal linker and ligation procedure for construction of genomic DNA libraries enriched for microsatellites. Biotechniques 27(500–502):504–507 Hanaoka S, Muturi G, Watanabe A (2012) Isolation and characterization of microsatellite markers in Melia volkensii Gurke. Conserv Genet Res 4(2):395–398 Untergasse A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen AM (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:71–74. http://www.bioinformatics.nl/cgibin/primer3plus/primer3plus.cgi Winters G, Shklar G, Korol L (2013) Characterizations of microsatellite DNA markers for Acacia tortilis. Conserv Genet Res 5:807–809
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