Conservation Genet Resour (2011) 3:441–444 DOI 10.1007/s12686-010-9375-2
TECHNICAL NOTE
Isolation and characterization of novel microsatellite loci in Vaccinium arctostaphylos L Sepehr Mohajeri Naraghi • Mohsen Mardi • Tahereh Hasanloo • Seyed Mostafa Pirseyedi Parvaneh Mahmoodi
•
Received: 8 December 2010 / Accepted: 15 December 2010 / Published online: 1 January 2011 Ó Springer Science+Business Media B.V. 2010
Abstract For the first time, this study has described the characterization of 25 novel polymorphic microsatellite loci from a repeat-enriched genomic library of V. arctostaphylos using a modified FIASCO method. Polymorphism of each locus was assessed in 64 individuals of the Vaccinium. The average allele number of the microsatellites, observed (HO) and expected (HE) heterozygosities were 5.88, 0.85 and 0.67 per locus, respectively. The polymorphic information content (PIC) value and inbreeding coefficient (F) ranged from 0.26 to 0.87 and -0.72 to 0.03, respectively. Ten of the twenty-five loci showed significant departures from Hardy–Weinberg equilibrium. These microsatellite loci could be useful to study genetic diversity and population structure of V.arctostaphylos. Keywords Microsatellite marker FIASCO Genetic diversity Vaccinium arctostaphylos L
Vaccinium arctostaphylos L. (Caucasian whortleberry) is a perennial and tetraploid (2n = 4x = 48) plant which grows in Iran, Turkey, Armenia, Azerbaijan, Georgia,
S. Mohajeri Naraghi M. Mardi (&) S. M. Pirseyedi P. Mahmoodi Department of Genomics, Agricultural Biotechnology Research Institute of Iran (ABRII), Mahdasht Road, P. O. Box: 31535-1897, Karaj, Iran e-mail:
[email protected] URL: http://www.abrii.ac.ir T. Hasanloo Department of Molecular Physiology, Agricultural Biotechnology Research Institute of Iran (ABRII), Mahdasht Road, Karaj, Iran
Russia and Bulgaria (Darrow et al. 1944; Davis 1965–1988; Facciola 1990; Huxley 1992; Komarov et al. 1934–1964; Kru¨ssmann 1984–1986). The plant is widely used in Iranian folk medicine as an antidiabetic and antihypertensive agent (Sedaghathoor 2007). There is no report about the genetic diversity of V. arctostaphylos. It is essential to use molecular markers as genetic tools for further exploration of V. arctostaphylos also as powerful tools for the conservation genetics and population structure. In this report the characterization of 25 new polymorphic microsatellite loci in V. arctostaphylos has been described. The total genomic DNA was extracted from young leaves of V. arctostaphylos by using the Plant Genomic DNA Isolation Kit (Core Bio System, Korea). A SSRenriched library was constructed from the DNA of four accessions of four different populations by using the protocol of Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) (Zane et al. 2002), with some modifications. The DNA (250 ng of each one) was first digested with the restriction MseI (Fermentas, Germany). Following that ligated to MseI adapters (MWG Biotech, Germany). The digestion-ligation fragments were amplified in two stages (pre-amplification and selective amplification) by PCR. The processes of the SSR isolation from the beginning to the final step of the pre-amplification stage were separately conducted for the DNA of each accession, but the PCR products of all four accessions from the preamplification stage were mixed and used for the selective amplification. The PCR products were denatured and hybridized to twelve different 50 biotinylated probes based on di- and tri-nucleotide repeats [(GC)17, (AC)17, (CT)17, (AT)17, (GT)17, (AG)17, (ACC)10, (ACT)10, (GTT)10, (GGT)10, (ATT)10 and (CTT)10] (MWG Biotech, Ebersberg, Germany). The DNA molecules attached to the
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Genbank accession no.
HQ013263
HQ013264
HQ013265
HQ013266
HQ013267
HQ013268
HQ013269
HQ013270
HQ013271
HQ013272
HQ013273
HQ013274
HQ013275
HQ013276
HQ013277
HQ013278
HQ013279
Locus
ABRII-VA-2
123
ABRII-VA-6
ABRII-VA-10
ABRII-VA-11
ABRII-VA-14
ABRII-VA-15
ABRII-VA-16
ABRII-VA-17
ABRII-VA-18
ABRII-VA-19
ABRII-VA-20
ABRII-VA-21
ABRII-VA-22
ABRII-VA-24
ABRII-VA-27
ABRII-VA-29
ABRII-VA-30
R: GAAAAAAAACCAACAACAG
F: TCATCGTCTCTCACTCTCCC
R: AGGGAGAGTGAGAGACGATG
F: GAATCCAGACAAGTCCAAGC
R: AATAATGATGGTGTTGGTGG
F: TCATTCCTCCTCCTATTTCTG
R: AGATACGTTGGCTGTGTTCG
F: ACGTACCAAATTAGGATGCG
R: AGTCCTGAGTAATAACGGCG
F: CTTTTGCTTTATTTCCCTCC
R: ATATCATAGGGAATCAGGCG
F: GTATTCGACCAGCATTATGTG
F: TTTGATTATGGGTTGGTTTG R: AAACCTGTACACATCTCGGG
R: GTTGGCTTGATCTTTTTCCC
F: GCTTCCATCCTCACCTTGTC
R: AAACCATTGGGAAATATGCC
F: GGGAAAAAGATCAAGCCAAC
R: AATGTGGAACTAAAATGGCC
F: TAAGCAACCTGACACGAAGC
R: AGGGATGCAATTCAAGTGTG
F: CTTGCTACGATGACTACGCC
R: ATTGCATCCCTTTCTTCCAG
F: AAGCCGAAGCAACAGACTC
R: TCCCTCTGTTTTCAATACGG
F: GATCTGGGTTTGGTGAACAC
R: TCGATCAGCTTAGGTGTTGTG
F: CAAACAAGCCCAAAATAGA
F: TCATCTTTTTTGTTCACCCC R: TCTATTTTGGGCTTGTTTGG
R: GAGTCCTGAGTAAACAGCCG
F: CATAATAGGGGTTGCTTTCG
R: AGCAGAGGTTTGTTTGATTG
F: TAAATTTCTCGGCCTCTCAC
Primer sequence (50 –30 )
(TC)12 (AC)10
(CTT)8 (TC)9
(GAAA)4
(TC)15 (CAAAA)3
(AG)11
(CAAGGA)2
(TTATTT)2
(AGCAGT)2
(AATATG)2 (GGCATA)2 (AT)4
(TCAA)3
(TGATCA)2
(AC)9 (CT)2 (CT)6
(GA)7 (AG)19
(TTTTTTTA)2 (ATTT)5
(TGTT)3 (GT)2 (GGT)3
12
11
3
7
5
5
4
3
2
3
3
5
17
5
2
6
10
(TCGGTGG)2 (GA)16 (TTC)2 (TTC)5 (TCCCTC)2
No. of alleles
Repeat motif
Table 1 Characterization of 25 polymorphic microsatellite loci in Vaccinium arctostaphylos
143–165
143–173
166–174
213–243
273–281
162–186
117–134
220–244
207–219
208–220
138–150
229–237
222–252
170–186
169–177
200–230
191–209
Allele size range (bp)
0.98
0.94
0.98
0.81
0.89
0.78
0.92
1
0.41
1
0.91
0.64
0.94
0.84
0.55
0.55
0.85
HO
0.88
0.79
0.66
0.77
0.62
0.62
0.6
0.58
0.3
0.64
0.65
0.68
0.86
0.74
0.38
0.61
0.83
HE
0.87
0.77
0.59
0.74
0.55
0.56
0.52
0.49
0.26
0.56
0.58
0.62
0.84
0.7
0.31
0.58
0.8
PIC
-0.09
-0.11
-0.19
-0.06
-0.17
-0.11
-0.2
-0.27
-0.09
-0.22
-0.15
-0.4
-0.08
-0.08
-0.12
0.01
-0.04
r
-0.19*
-0.27*
-0.49
-0.15*
-0.44
-0.28
-0.54
-0.72
-0.38
-0.57
-0.39
-0.12*
-0.18*
-0.19*
-0.43
0.31*
-0.1*
F
442 Conservation Genet Resour (2011) 3:441–444
HQ013280
HQ013281
HQ013282
HQ013283
HQ013284
HQ013285
HQ013286
HQ013287
–
ABRII-VA-32
ABRII-VA-33
ABRII-VA-34
ABRII-VA-36
ABRII-VA-37
ABRII-VA-38
ABRII-VA-39
ABRII-VA-40
Mean
–
R: GTTTTTTGGGTACCGTTGAG
F: GACTGCAAGCTCCAAGAAC
R: TTATCAGACAGTTGGTGGG
F: TCTTGCTTGAACTTCTCGTG
R: CACGAGAAGTTCAAGCAAG
F: ATCAATACTCTGCCTCTGCC
R: GCAGAAGAGGAAGTAGTCG
F: CACAACTGCGTCAGATTCTC
F: TTAGCCAAAGCAACCATTAG R: AGAATCTGACGCAGTTGTG
R: CATCCTCTTTGACAGCACTC
F: TTGCAGGTGTACACTTCAGG
R: CATTGCCTTTGGATCAGTAG
F: GCTGCTCGTTCACTTGTTAC
R: TACGCATCTTTTTGGTGAAC
F: ATAAGAAAGAATGTGTGCGC
Primer sequence (50 –30 )
–
(TC)13
(TTTTTCT)2
(CT)18
(GGT)4 (GAGG)4
(GTTTTGT)2 (GTTTTGT)2
(ATG)4
(TG)6 (TGAG)5
(AG)14
Repeat motif
5.88
5
4
5
4
4
4
9
9
No. of alleles
–
255–263
266–287
254–262
208–220
150–171
171–180
213–253
149–165
Allele size range (bp)
0.85
0.88
0.84
0.89
0.95
1
0.94
0.86
0.93
HO
0.67
0.72
0.62
0.68
0.63
0.73
0.64
0.8
0.82
HE
0.62
0.68
0.55
0.63
0.55
0.68
0.57
0.78
0.79
PIC
-0.13
-0.13
-0.14
-0.17
-0.2
-0.15
-0.18
-0.06
-0.09
r
-0.33
-0.33
-0.36
-0.43
-0.52
-0.36
-0.47
-0.15*
-0.2*
F
HO Observed heterozygosity, HE Hardy–Weinberg expected heterozygosity, PIC Polymorphism information content, r Null allele frequency, F Inbreeding coefficient, * Indicates significant deviation from Hardy–Weinberg equilibrium (HWE) (P \ 0.05)
Genbank accession no.
Locus
Table 1 continued
Conservation Genet Resour (2011) 3:441–444 443
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biotinylated probes were captured using Streptavidin-conjugated magnetic beads (BioMagÒ; Qiagen, Germany). The non-specific DNA was removed by four non-stringency (10 mM Tris–HCl, 1 mM EDTA, 1 M NaCl) and three stringency (0.29 SSC and 0.1% SDS) washes and the DNA was recovered with magnetic field separation. The recovered DNA fragments were amplified by PCR using the pre and selective amplifications. The PCR products were ligated into pGEM-T Easy vector (Promega, Germany), and transformed into Escherichia coli DH5a following the manufacturer’s instructions. Recombinant clones were identified by blue/white screening and restriction enzyme EcoRI analysis (Fermentas, Germany). Among the 360 candidate clones, 55 were purified using a plasmid extraction kit (Core-Bio, Korea) and sequenced (Macrogen Sequencing Service, South Korea). A total of 31 (57%) sequences were found to contain the flanking regions of the SSRs using SSR Locator software (Maia et al. 2008), of which 43 primers were designed with the software Oligo ver. 4.04 (Rychlik 1992). Polymorphisms of all 43 microsatellite loci were assessed in 64 individuals of V. arctostaphylos from four geographically separated populations of Iran. The PCR reactions were performed in an ABI thermal cycler in 15 ll of reaction containing 20 ng DNA, 1.5 ll 19 PCR buffer,1.2 ll of 2 mM MgCl2, 1 ll of 10 mM dNTPs, 0.9 ll of each primer (10 pmol/ll), and 0.5 U Taq DNA polymerase. PCR amplifications were conducted under the following conditions: 5 min at 95°C, followed by 10 touchdown cycles of 30 s at 95°C, 45 s at 63°C (1°C lower per cycle) and 40 s at 72°C, and 25 cycles of 30 s at 95°C, 30 s at 53°C and 40 s at 72°C, with a final extension step of 7 min at 72°C. PCR products were separated on 6% denaturing polyacrylamide gel using a 50 bp DNA ladder by silver staining. Due to the tetraploidy of V. arctostaphylos, statistical calculations were performed using the TETRA program (Puyvelde et al. 2010). The number of alleles per locus was assessed. Hardy– Weinberg expected heterozygosity (HE) and Observed heterozygosity (HO) were evaluated (Nei 1978). Polymorphism information content (PIC) was estimated (Botstein et al. 1980). Inbreeding coefficient or fixation index (F) was calculated (Berg and Hamrick 1997). Frequency of null alleles (r) was estimated (Brookfield 1996). The loci were tested for Hardy–Weinberg equilibrium (HWE) using a simple X2 contingency table which is reformulated in terms of the fixation index (F) and significance levels were determined (Li and Horvitz 1953). Overall, 25 out of the 43 primer pairs (59%) displayed polymorphisms (Table 1). The number of alleles varied from 2 to 17 per locus and the values observed (HO) and expected (HE) heterozygosities ranged from 0.41 to 1 and from 0.30 to 0.88, respectively (Table 1). The mean PIC value across all loci was 0.62. According to the criterion defined by Botstein
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Conservation Genet Resour (2011) 3:441–444
et al. (1980), of the 25 loci, 22 were highly informative (PIC [ 0.5) and three were reasonably informative (0.5[ PIC [0.25). The average inbreeding coefficient (F) was -0.33 (Table 1). The results showed that ten of twentyfive polymorphic loci deviated from Hardy–Weinberg equilibrium (PHWE \ 0.05), probably due to the small sample size, genetic drifting and the presence of null alleles. Conductively, these 25 polymorphic microsatellite markers have the potential for use as genetic markers in areas such as genetic diversity, conservation genetics, population structure and molecular identification of Vaccinium arctostaphylos. Acknowledgments We gratefully thank Ms. Rooshanak Sepehrifar for providing us with the plant materials. This study was supported by Agricultural Biotechnology Research Institute of Iran (ABRII).
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