Beruflich Dokumente
Kultur Dokumente
DOI 10.1007/s12686-014-0361-y
MICROSATELLITE LETTERS
Introduction
Dactylorhiza hatagirea (D. Don) Soo, an orchid of high
economic importance is endemic to western Himalayan
region (2,5005,000 m asl). It is an erect herb with
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F-AAACAAACATGCCCCAGTTA
F-GGTGTTCCTAACTGCCCACT
F-TCAGCGGAGGAGAGGTAGAA
R-CTCCTCGCGAATGAAATGAT
F-TAAACAAACATGCCCCAGTT
R-GAGCCGGACATGAGAGTTTC
F-CATGCCCCAGTTATCCACTT
R-TGGCCACTTGTAGTGAGCTG
(TA)15
(TA)8
(GAA)6
50
53
56
53
54
56
51
52
50
53
53
53
51
50
53
Ta (C)
190205
210230
230360
390405
550570
130150
190200
230
150320
320380
205400
360480
205220
150250
200290
4.2
No. of
alleles
0.623
0.500
0.100
0.400
1.00
0.600
0.400
0.400
0.800
0.800
0.625
0.900
0.900
0.500
0.800
Ho
0.631
0.789
0.658
0.337
0.611
0.442
0.337
0.353
0.774
0.858
0.892
0.763
0.813
0.394
0.821
He
0.051
0.000*
0.502
0.029*
0.223
0.502
0.929
0.046*
0.013*
0.007*
0.029*
0.164
0.354
0.001*
0.532
0.709
0.551
0.269
0.492
0.332
0.269
0.303
0.685
0.790
0.816
0.680
0.504
0.305
0.751
PIC
DQ022920
DQ022926
JQ229975
GQ244815
EU176076
DQ986357
DQ986360
DQ986361
EU884288
AY283526
AY283531
AY283532
DQ022918
EF594447
AY283535
Gene bank
accession no.
Gene bank accession no. of the sequence from which primers were designed
Ta annealing temperature, Ho observed heterozygosity, He expected heterozygosity, P probability that genotype proportions conform to HardyWeinberg equilibrium, PIC polymorphism
information content
Mean
KSSR-39
KSSR-37
KSSR-35
(CAA)3
KSSR-32
R-TGGGACTCTCTCTTTATTCTCGTC
(TA)8
F-GCCCGCGAACACTTTATTTA
(TCT)8
(AGA)6
(GAA)6
(TA)6
(TTC)4
R-CTCCTCGCGAATGAAATGAT
F-CGATGGAAGCTGTTCTAACGA
R-GACTGCAGGTAAGGGCTCAG
F-AAGGTACCACGCTTCGTCAG
R-CTCAATCATCCAAAGGGACAA
F-CTGGAAGTAGGGGGAGCAAT
R-CGATCCTCATCCTGTTTTGC
F-CGCCGACAAACTCTACATCG
R-GGGAAATGAACCTTTTGCAC
F-CGCGAAGTCAAGATTGAAAA
R-GAGAAAGAACGCCAAAGACG
(AGA)3
(TTC)4..(TTCCTC)3
(TA)6
(TA)6
(TTC)4
Repeat motif
KSSR-30
KSSR-22
KSSR-21
KSSR-20
KSSR-18
KSSR-15
F-CAGGGGGATAAGTTCTCGAC
KSSR-12
R-AGAAAGAACGCCAAAGACGA
F-TCCTCTGCAGTCTTGTTCCA
R-GAGAAAGAACGCCAAAGACG
R-GAGCCGGACATGAGAGTTTC
KSSR-11
KSSR-07
R-CCCGGCCAGTACTTAACCAG
F-CGCGAAGTCAAGATTGAAAA
R-AGAAAGAACGCCAAAGACGA
F-GGTCCAGGGGGATAAGTTCT
KSSR-02
KSSR-04
Marker name
72 C and final extension for 7 min at 72 C. The amplicons were separated on 6 % denaturing polyacrylamide
gels in 19 TBE buffer and visualized by silver staining.
Polymorphic alleles for each marker were scored manually
and the data were utilized for computing various diversity
indices. Expected heterozygosity (He), Observed heterozygosity (Ho) and HardyWeinberg equilibrium were
obtained using POPGENE version 1.32 (Yeh and Boyle
1997) and polymorphism information content (PIC) was
calculated as per Botstein et al. (1980).
From the 784 nucleotide sequences, 35 primer pairs
flanking microsatellite motifs were designed. Out of the
primer pairs tested across a panel of 20 D. hatagirea ecotypes, only 15 amplified unambiguous amplicons. Of the 15
markers giving reliable amplification, 14 were polymorphic
(Table 1). These 15 primers amplified 64 alleles with a
mean value of 4.2 alleles in a size range of 130570 bp. Ho
and He values for polymorphic loci varied from 0.100
(KSSR-37) to 1.000 (KSSR-32) and 0.337 (KSSR-35) to
0.892 (KSSR-12) with average of 0.623 and 0.631,
respectively. Minimum PIC value of 0.269 was recorded
for the markers KSSR-22 and KSSR-35, whereas a maximum PIC value of 0.816 was recorded for KSSR-12 with
an overall mean PIC of 0.532. Seven markers significantly
deviated from HardyWeinberg equilibrium at P value of
0.005. These values of diversity indices clearly indicated
their potential for uncovering the genetic diversity of D.
References
Bhatt A, Joshi SK, Gairola S (2005) Dactylorhiza hatagirea (D. Don)
Sooa west Himalayan orchid in peril. Curr Sci 89(4):610612
Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of
a genetic linkage map in man using restriction fragment length
polymorphisms. Am J Human Genet 32:314331
Lin P, Zeng L, Yang Z, Liu R, Zhong Y (2014) Development and
characterization of polymorphic microsatellitemarker for Dactylorhiza hatagirea (D. Don) Soo. Conserv Genet Res. doi:10.
1007/s12686-013-0053-z
Sharma V, Bhardwaj P, Kumar R, Sharma RK, Sood A, Ahuja PS
(2009) Identification and cross-species amplification of EST
derived SSR markers in different bamboo species. Conserv
Genet 10:721724
Yeh FC, Boyle TJB (1997) Population genetic analysis of codominant and dominant markers and quantitative traits. Belg J
Bot 129:157
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