Online Resource 3
(a) Development of microsatellite markers for Abies mariesii
Microsatellite marker was developed using an improved technique for isolating codominant compound microsatellite markers
(Kaneko et al. 2008; Lian and Hogetsu 2002; Lian et al. 2006). Total genomic DNA of A. mariesii was extracted from leaves using a
modified CTAB method (Murray and Thompson 1980). An adaptor-ligated, restricted DNA library of A. mariesii was then
constructed according to the following procedure: DNA was digested with the blunt-end restriction enzyme, Afa I. The restriction
fragments were then ligated with a specific blunt adaptor (consisting of the 48-mer: 5’GTAATACGACTCACTATAGGGCACGCGTGGTCG ACGGCCCGGGCTGGT-3’ and an 8-mer with the 3’-end capped with an
amino residue: 5’-ACCAGCCC-NH2-3’) using the DNA ligation kit (Takara). Fragments were amplified from the Afa I DNA library
using compound SSR primer (AC)6(AG)7, (AG)6(AC)7 or (AC)6(TC)7 and an adaptor primer (5’-CTATAGGGCACGCGTGGT-3’).
The amplified fragments, ranging from 400 to 800 bp, were then separated on a 1.5% LO3 agarose gel (Takara) and purified using the
QIAquick Gel Extraction Kit (Qiagen). The purified DNA fragments were subsequently cloned using the QIAGEN PCR Cloning plus
Kit (Qiagen) according to the manufacturer’s instructions. In brief, polymerase chain reaction products were ligated into the pDrive
vector, and transformed into QIAGEN EZ competent cells. Transformants were identified by blue/white screening on LB agar plates
containing ampicillin, X-gal and IPTG. The cloned fragments were amplified using the M13 forward and reverse primers from the
plasmid DNA of positive clones. Amplified fragments were sequenced using the BigDye Terminator Cycle Sequencing Kit (Applied
Biosystems) and ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). For each fragment containing (AC)6(AG)5, (AG)6(AC)5
or (AC)6(TC)5 compound SSR sequence at one end, a specific primer was designed from the sequence flanking the compound SSR
using Primer3 software (Version 0.4.0, Rozen and Skaletsky 2000).
(b) Characteristics of six microsatellite loci for A. mariesii
Locus
Am003
DDBJ accession no.
AB911247
Size Range (bp)
191-235
Repeat motif
(AC)6(AG)22
Am247
AB911250
110
(AC)6(TC)7
Am351
AB911248
133-157
(AG)6(AC)17
Am364
AB911249
126-140
(AG)6(AC)9
Am418
AB911252
215-269
(AC)6(AG)26
Am548
AB911251
118-132
(AC)6(TC)7
Primer sequence (5'–3')
ACACACACACACAGAGAGAGAG
AATGTGGAATGCTTAAAGTGAA
ACACACACACACTCTCTCTCTC
TTATTCTGTCTTAAGCATTTTAGTTCT
AGAGAGAGAGAGACACACACAC
GCCTATTGGCAAAAGCTTAG
AGAGAGAGAGAGACACACACAC
GAACCCGAGTAGGGGATG
ACACACACACACAGAGAGAGAG
CAACTGGTTTGTGCTTGATT
ACACACACACACTCTCTCTCTC
AGCCATTGAAGGATTTACTGA
Ta (℃)
57
HO
0.932
HE
0.865
57
0.000
0.000
57
0.715
0.693
57
0.291
0.326
57
0.890
0.901
57
0.464
0.509
Ta, annealing temperature of primer pair; HO, observed heterozygosity; HE, expected heterozygosity. HO and HE were summarized over
all 179 samples for each locus. DDBJ accession numbers are available to the public after manuscript being accepted for publication.
(c) PCR amplification mixtures and thermal profile used for genotyping A. mariesii samples
PCR amplification mixtures were prepared in a final volume of 6.0 μl, which contained 2.13 μl of ultrapure water, 0.06 μl of
each 20 μM forward and reverse primer, 3 μl of Multiplex PCR Master Mix (Qiagen) and 0.75 μl of DNA template (37.5-75 ng). The
PCR thermal profile involved an initial denaturation step at 95 °C for 15 min followed by 30 cycles at 94 °C for 0.5 min, 57 °C for 1.5
min and 72 °C for 1.0 min, with a final extension at 60 °C for 30 min.
References
Kaneko S, Franklin D, Yamasaki N, Isagi Y (2008) Development of microsatellite markers for Bambusa arnhemica (Poaceae: Bambuseae), a
bamboo endemic to northern Australia. Conserv Genet 9:1311-1313
Lian C, Hogetsu T (2002) Development of microsatellite markers in black locust (Robinia pseudoacacia) using a dual-supression-PCR technique.
Mol Ecol Notes 2:211-213
Lian CL, Wadud MA, Geng Q, Shimatani K, Hogetsu T (2006) An improved technique for isolating codominant compound microsatellite
markers. J Plant Res 119:415-417
Murray MG, Thompson WF (1980) Rapid isolation of high molecular-weight plant DNA. Nucleic Acids Research 8:4321-4325
Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds)
Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386