Ap Applicatitions Ons: L L, Z - F W, Q - M L, W - H Y, H - L C, Z - M W
Ap Applicatitions Ons: L L, Z - F W, Q - M L, W - H Y, H - L C, Z - M W
Ap Applicatitions Ons: L L, Z - F W, Q - M L, W - H Y, H - L C, Z - M W
Applicati
Ap
tions
ons
in Pl
Plantt Scien
Sciences
ces
PRIMER NOTE
Premise of the study: Although there are as many as 250 species in the genus Beilschmiedia, their genetic diversity has been
poorly investigated. Our objective was to develop microsatellite markers for B. roxburghiana to study its genetic diversity for
the sustainable management of this species.
Methods and Results: Using the microsatellite-enriched library and PCR-based screening method, 22 microsatellite markers
were developed and 10 showed high polymorphism in a population. The number of alleles per locus for these 10 microsatellites
ranged from five to 19. The observed and expected heterozygosities ranged from 0.298 to 1.000 and from 0.314 to 0.878,
respectively.
Conclusions: Our results from the 10 highly polymorphic microsatellites indicate that the principal reproductive mode of
B. roxburghiana is clonal in the studied population. These microsatellites will facilitate further studies on genetic diversity and
structure in B. roxburghiana.
Key words:
banna Plot.
Beilschmiedia roxburghiana; genetic diversity; Lauraceae; microsatellite markers; population genetics; Xishuang-
doi:10.3732/apps.1200549
Applications in Plant Sciences 2013 1(8): 1200549; http://www.bioone.org/loi/apps 2013 Botanical Society of America
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repeats in 208 sequences. We then used Primer3 software (Rozen and Skaletsky,
2000) to design primers for these sequences, of which 66 were discarded
because repeats were too close to one end of the sequences or because Primer3
found no suitable primers. PCR amplifications were performed for the remaining
142 sequences in a 20-L volume containing 20 mM Tris-HCl (pH 8.4), 100 mM
(NH4)2SO4, 3 mM MgCl2, 0.4 mM dNTPs, 0.4 M each primer, 50 ng of
genomic DNA, and 1 U Taq polymerase (TaKaRa Bio Inc.). The amplification
program was 95C for 5 min, 35 cycles of 94C for 30 s, optimized annealing
temperature for 30 s, and 72C for 45 s, with a final extension at 72C for 10 min.
We checked the PCR products on 2% agarose gels and found that 41 of them
could be successfully amplified with expected sizes.
Polymorphisms were initially assessed in these 41 microsatellite loci using
12 individuals that were randomly selected from 421 samples of B. roxburghiana collected from the Xishuangbanna Plot in the Mengla Nature Reserve
(21364258N, 101342647E). PCRs were performed using the same
procedure described above. Electrophoresis of the products was done on an
ABI 3730 sequencer (Applied Biosystems, Carlsbad, California, USA), and
fragment lengths were analyzed using ABI GeneMapper software version 3.7
(Applied Biosystems).
TABLE 1.
Locus
Repeat motif
BR01
(TG)17(AG)14
BR02
(CAG)4(GA)14
BR03
(AG)9TAAG(GA)11
BR04
(TC)16
BR05
(AC)18(AG)9AAG
BR06
(GA)18
BR07
(CT)9
BR08
(TC)20
BR09
(GA)21
BR10
(AG)10AA(AG)3A4TACGT(GA)5
BR12*
(CT)9C(A)27
BR13*
(AC)14
BR15*
(AG)16
BR17*
(AG)15
BR20*
(AG)7
BR21*
(AG)9
BR11
(TG)7(CG)6(TG)3
BR14
(TC)17
BR16
(AG)7AAATCTTGA(AG)6
BR18
(GA)9
BR19
(TC)8
BR22
(TC)7
ATGGGAATATCGCATCAT
GTTACCAGACTTGGAGCA
AACCGTTATCTGGACATTG
TTCGCCTTCCTTCTTTTC
CATTTCCGTCAATGCCTGAT
ACCATTTCAATGGCGAAGAG
CTCCAGTCCACGGCAATC
GACCAAACCTGAACCTACAA
CAATTCAAATGAAAGCGACTGA
TCAGACCCAAAGGGAGACAC
GTCGGAGATTCAAACATA
AGTGGGATTACCAAGAGG
GCTGCTGTTTCCTTGGACTT
CCTGGGTTCTCTCAGCTTTG
GAGACGCGTTTTCTCTCTCG
GGGATTTTGCAAACCTGAGA
TTTTGGCAACAACAGATCCA
TGCAAAATTCGTGCATTGAT
TATTGATTCCGAGGCTTTCG
CCCCACCATCACCTCTATTTT
GTGAAGTATGCAGTATAAAAGG
GATTGAGCAGAGGGTGTT
TGTATGCGTGTATGCTGT
TCACTTCTATTCGGTTTCA
ATGCGAGCCACTTGTGAAG
CATCTCCATCCCCCTTCTCT
AGAAACGACCCAGTCCCA
CAGGTAAGGCCCAACCGA
ACCTGCAATGGTGCCAAATA
CATCACACCCACTGCAAAAC
AAGGAAGGTGTTAGGGTT
GTTTACAAAAGATGAAAGGA
TTGCGTATCTTGCACATC
CACTTACTCACCACTCCTTTA
TATACCAGACCCTATTAGCC
CCAACTGCCTGTTTCATT
GAAGGCACTGGGAACTCT
GCAATCTGCTGAAGGGAT
TACGCGTACAAAGGGGTAGG
CAGTCGTCTGCCCTCATACA
AGTCGATCCCCTCTTTCCTC
GCAGCAATGCTTGTTTGAGA
GGAAGAAAGCCCCTAAAT
CACAGTCCACGACAGTAATC
Ta (C)
5-fluorescence label
GenBank
accession no.
101115
50
FAM
JX524138
131149
50
FAM
JX524139
183195
50
FAM
JX524140
177220
50
FAM
JX524141
212266
50
HEX
JX524142
166204
52
FAM
JX524143
174182
52
FAM
JX524144
135178
55
FAM
JX524145
161185
55
FAM
JX524146
221241
55
HEX
JX524147
158164
50
FAM
KC211772
200206
50
HEX
KC211773
230247
52
FAM
KC211775
161169
55
FAM
KC211777
234238
55
FAM
KC211780
300310
55
HEX
KC211781
150
50
FAM
KC211771
243
50
HEX
KC211774
166
55
FAM
KC211776
172
55
FAM
KC211778
214
55
HEX
KC211779
326
55
HEX
KC211782
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TABLE 2.
Locus
Ho
He
BR01
BR02
BR03
BR04
BR05
BR06
BR07
BR08
BR09
BR10
Mean
8
7
7
19
15
17
5
14
13
13
0.729
0.567
0.298
0.995
1.000
0.993
0.882
0.911
1.000
0.979
0.834
0.655
0.666
0.314
0.807
0.798
0.878
0.584
0.816
0.820
0.804
0.713
0.113
0.149*
0.049*
0.233*
0.254*
0.131*
0.511*
0.116*
0.220*
0.218*
0.170*
CONCLUSIONS
Twenty-two microsatellites of B. roxburghiana were isolated
and tested. Our data indicate that 10 are highly polymorphic. A
large negative fixation index (F) estimate (0.170, Table 2), the
presence of many identical genotypes among the individuals,
and the presence of significant multilocus linkage disequilibrium suggest that the principal reproductive mode of B. roxburghiana may be clonal for this plot, which is unexpected.
Further studies are needed to confirm this.
In the future, the microsatellites developed in this study will
be useful to investigate the effects of habitat fragmentation on
APPENDIX 1.
Voucher no.
Collector name
Collection locality
109113
107464
Shi-Shun Guo
Wang Hong
http://www.bioone.org/loi/apps
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