Conservation Genetics Resources
Electronic Supplementary Material
ESM_1. Additional details including an extended, quantitative descriptions of clone
screening and 454-sequencing results, multiplex groups and the assignment of South
Island robin microsatellite loci to homologous locations in the zebra finch genome.
Isolation and characterisation of microsatellite markers from the South Island
robin (Petroica australis)
Sheena M. Townsend, Tania M. King and Ian G. Jamieson
Allan Wilson Centre for Molecular Ecology and Evolution, Department of Zoology, University of
Otago, 340 Great King Street, Dunedin 9016, New Zealand
townsend.sheena@gmail.com
Enriched clone screening
Overall, 942 colonies were screened for repeats resulting in 67 (7.1%) positive
clones. Of these, 39 were sequenced using an ABI 3730 Genetic Analyser (Applied
Biosystems). Sequences were edited and checked for duplicates using SEQUENCHER
v3.7 and 21 pairs of primers designed using PRIMER3PLUS
(http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) (Untergasser et
al. 2007)
454 sequencing details
Our sequencing run was scaled to 1/8th of a plate on the Roche 454 GS-FLX
System (Roche, Penzberg, Germany. This yielded 42,098 fragment reads (7.3Mb
sequence overall) with an average read length of 174bp. Reads were screened for
microsatellite repeats using MSATCOMMANDER v0.8.1 (Faircloth 2008) to locate di(194), tri- (287) and tetranucleotide (112) repeat regions. Of these, 54 di-, 111 tri- and
33 tetranucleotide repeat-containing reads had sufficient flanking sequence to allow
primer design. After eliminating potential duplicates using SEQUENCHER v3.7 we
designed 48 primer pairs using PRIMER3PLUS (Untergasser et al. 2007).
Location of loci in the zebra finch genome
To ensure that microsatellite loci did not represent duplicates, we conducted a
BLAST search using NCBI’s BLAST 2 Sequences protocol with the sequences upon
which primer development was based included as both reference and searched
sequences (Dawson et al. 2006; Olano-Marin et al. 2010). All matches with scores
below 50 and E-values less than
were ignored. This search indicated no
matches between pairs of loci.
To determine the zebra finch (Taeniopygia guttata) chromosome locations of
loci, we followed the methods of Olano-Marin et al. (2010) see also Dawson et al.
(2006). VecScreen (http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html UniVec
build 6.0) revealed no vector contamination in any sequences. We initially searched
clone and 454 sequences using BLAT at the UCSC Genome Bioinformatics website
(http://genome.ucsc.edu/) with the Zebra finch Jul. 2008 (WUGSC 3.2.4/taeGut1)
assembly. Positions were confirmed using BLASTN 2.0MP-WashU (WUSTL)
(http://genome.wustl.edu/tools/blast) and the Taeniopygia guttata–3.2.4chromosomes database with DUST/SEG filter and RepeatMasker.
We followed Olano-Marin et al. (2010) in our criteria for non-ambiguous
location assignment, accepting matches with an E-value less than
. In the
case of multiple matches, if the second best match was more than an order of
magnitude weaker, we accepted the best match. When the top two matches included
the unknown chromosome, we used the named chromosome assignment if the above
criteria were met. When these criteria were not met or when BLAT and BLAST did
not agree, we searched for sequences using the reference genome at NCBI
(http://www.ncbi.nlm.nih.gov/blast). In all cases, except one, this confirmed
assignment of the chromosome from the initial BLAST (WUSTL) search (Table S1).
We were not able to unambiguously match the locus Pau24 to a homologous
position in the zebra finch genome. We further extended our attempt to do so by
searching for the Pau24 sequence within zebra finch WGS reads using the NCBI
BLAST search. This also failed to return any well-supported, unambiguous matches.
Pau8 and Pau26 were assigned unambiguously to chromosomes via both
BLAST searches (WUSTL and NCBI) however, BLAT returned ambiguous start
positions for these loci. No location was well-supported compared to other potential
locations and the top choices did not correspond to confirmed chromosomal matches,
therefore we searched NCBI WGS reads and conducted a BLAT search on the top
matching homologous zebra finch sequence to find the best supported approximate
start locations for these loci. For a third locus, Pau77, the top start position
assignment in BLAT was only weakly preferred over other choices, but consistent in
terms of chromosome assignment with both BLAST servers. Conducting a BLAT
search on the strongest homologous zebra finch sequence for this locus and
confirmed the initial location assignment, which was used.
Table S1. Supplementary details for 20 South Island robin microsatellite loci. For
amplification, loci were combined in multiplex PCR in three groups as indicated.
Locus
Repeat motif
Zebra finch
chr
Start
position
Multiplex
group
Pau1
Pau2
Pau4
Pau6
Pau7
Pau8
Pau9
Pau16
Pau17
Pau24
Pau25
Pau26
Pau28
Pau39
Pau63
Pau66
Pau67
Pau77
Pau81
Pau82
(TG)2 TTTG(TG)12TTTG
(AC)19 AG(AC)3
3
2891710
2
5
48006134
3
(AC)6 AA(AC)2 (AAC)2 AC(A)6
1A
54059184
2
(GT)10
1A
16985012
1
(GT)12
5
43897126
2
(TG)13 (TATG)4
4
39599233
1
(TG)22
5
984444
3
(TC)5 . . . (TC)2 TT(TC)3. . . (TC)6 A(AC)12
1A
27557900
1
(AC)7 . . . (TC)5
5
51323773
1
(AC)15
NA
NA
2
(TG)13
2
106694637
2
(GTT)12
1
8542288
3
(GT)8
13
16228286
1
(AG)7
5
57668372
1
(GGGA)6
2
39768919
2
(GACA)5
10
7612298
2
(AAAC)5
4
25406454
2
(TTTA)5
1A
54230324
1
(CCT)5
11
12181319
3
(GAG)6
1A
53337441
2
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