Additional File 1
BAC library construction
BAC library CM_MBc was constructed and the clones arrayed on Hybond N+
membranes (GE Healthcare Life Sciences, Piscataway NJ) as described for CMCMBb
and CMCMBd, except that the two rounds of size selection were performed in 1 x
TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0), using identical run conditions (5-15
s, 15 h, 14 C at 6 v/cm, 120 degree angle), and the slice of the first gel containing the
size-selected fragments was rotated 180° for the second round of size selection
(O’Neill and Bancroft 2000).
Probes and BAC library filter hybridizations
Molecular genetic markers were selected from all twelve chromosomes (Kubisiak et
al 1997, Sisco et al 2005, Paul Sisco, pers comm), including eight RFLPs, four
isozymes, cystatin, six simple sequence repeats (SSRs) (Barreneche et al 1998, Buck
et al 2003, Marinoni et al 2003), and four ESTs mapped in European chestnut
(Casasoli et al 2006) to linkage groups identified as homologous to linkage groups in
the map of the interspecific (Castanea mollissima x C. dentata) F2 population (Sisco
et al 2005) (Table A). Sequence of one of the RFLPs was provided by Robert
Bernatzky, who also supplied the clones of the remaining RFLPs for sequencing
(John E. Carlson, unpublished). The ExPASy website (http://enzyme.expasy.org/)
was used to identify Arabidopsis thaliana cDNA sequences corresponding to
isozymes phosphoglucose isomerase (PGI, EC 5.3.1.9), shikimate dehydrogenase
(SKD, EC 1.1.1.25), phosphoglucomutase (PGM, formerly EC 2.7.5.1, now EC 5.4.2.2),
and glutamate oxaloacetate transaminase, (GOT, EC 2.6.1.1); these sequences were
used in BLAST searches to identify corresponding chestnut cDNA sequences. Overgo
probes were designed from all of the chestnut sequences using overgo maker
(http://genome.wustl.edu/software/overgo_maker) or overgo (Cai et al 1998,
http://www.mouse-genome.bcm.tmc.edu/webovergo/OvergoInput.asp). Probes
were pooled in groups of ten or fewer for hybridizations to library filters, and
positive clones were rearrayed and probed individually as described in Horn et al
(2005). Overgo probes for SSR sequences QpZAG58, EMCs15, and CsCAT17
hybridized to more clones than was consistent with their expected copy number,
and the corresponding SSR primers (Barreneche et al 1998, Buck et al 2003,
Marinoni et al 2003) were used in PCRs to identify clones corresponding to the SSR
markers.
Table A. Molecular Probes hybridized to BAC library CM_MBc
Genetic
map LG
A
A
B
B
C
C
D
D
E
F
F
G
G
H
H
I
I
J
J
K
K
L
L
Integrated
Map LG
A
-B
-C
C
-I
E
-F
G
G
H
H
-I
-A
-J
L
--
Locus
CD192
PGI
CD175
SKD
GM49
PGM
CsCAT02
QpZAG58
CCMC39433
CD145
EMCs4
CD172
CCMC04326
cystatin
CD62
CD143
EMCs14
CD50
GOT
CsCAT17
CCMC09685
EMCs15
CCMC40039
Type of
marker
RFLP2
Isozyme
RFLP3
Isozyme
RFLP2
Isozyme
SSR
SSR
EST4
RFLP2
SSR
RFLP2
EST4
CAPS
RFLP2
RFLP2
SSR
RFLP2
Isozyme
SSR
EST4
SSR
EST4
No.
Physical map
Sequence1
BACs
ctg
CCall_contig40707_v2
14
11525
CCall_contig31965_v2
17
869
CCall_contig43518_v2
7
202
CCall_contig19078_v2
17
102
CCall_contig21087_v2
13
424
CCall_contig41632_v2
12
217
FJ868943
8
223
X98757
18
7995
CR627526
11 6310 (8773)5
CCall_contig16186_v2
13
2660
AJ505134
15
132
CCall_contig14665_v2
13 268 (11525)5
CF369280
14
5039
AF480168-AF480169
7
7166
CCall_contig39527_v2
18
2572
CCall_contig43303_v2
4
223
AJ505138
8
10757
CCall_contig2379_v2
10
1379
CCall_contig30441_v2
11
10889
FJ868954
14
263
CF369273
13 12028 (7679)5
AJ505139
20
1153
CR627835
8
4613
1Project
EST identifier or GenBank Accession number
obtained from John Carlson from clones supplied by Robert Bernatzky
3Sequence supplied by Robert Bernatzky
4EST markers mapped in European chestnut (Casasoli et al. 2006), on linkage
groups believed to be homologues of the ones listed in column 1 (Sisco et al 2005).
5Physical map contigs in parenthesis contain only one of the BACs positive for a
particular probe.
2Sequences
Acknowledgments
Construction and hybridization of BAC library CM_MBc was funded by grants from
the American Chestnut Foundation. The authors wish to thank Robert Bernatzky for
supplying RFLP sequence and clones, and Roberto Botta for providing SSR
sequences prior to their public release.
References
Barreneche T, Bodenes C, Lexer C, Trontin J-F, Fluch S, Streiff R, Plomion C, Roussel
G, Steinkellner H, Burg K, Favre J-M, Glössl, Kremer A (1998) A genetic linkage map
of Quercus robur L, (pedunculate oak) based on RAPD, SCAR, microsatellite,
minisatellite, isozyme and 5S rDNA markers Theor Appl Genet 97:1090-1103
Buck EJ, Hadonou, M, James CJ, Blakesley JD, Russell K (2003) Isolation and
characterization of polymorphic microsatellites in European chestnut. Mol Ecol
Notes 3:239-241
Cai W-W, Reneker J, Chow C-W, Vaishnav M, Bradley A. 1998. An anchored
framework BAC map of mouse chromosome 11 assembled using multiplex
oligonucleotide hybridization. Genomics 54:387-397
Casasoli M, Derory J, Morera-Dutrey C, Brendel O, Porth I, Guehl J-M, Villani F,
Kremer A (2006) Comparison of quantitative trait loci for adaptive traits between
oak and chestnut based on an expressed sequence tag consensus map. Genetics
172:533-546
Horn R, Lecouls A-C, Callahan A, Dandekar A, Garay L, McCord P,
Howad W, Chan H, Verde I, Ramaswamy K, Main D, Jung S, Georgi L, Forrest S, Mook
J, Zhebentyayeva TN, Yu Y, Kim HR, Jesudurai C, Sosinski BA, Arús P, Baird V, Parfitt
D, Reighard G, Scorza R, Tomkins J, Wing R, Abbott AG (2005) Candidate gene
database and transcript map for peach, a model species for fruit trees. Theor Appl
Genet 110:1419–1428
Marinoni D, Akkak A, Bounous G, Edwards KJ, Botta R (2003) Development and
characterization of microsatellite markers in Castanea sativa (Mill.) Mol Breeding
11:127-136
O'Neil CM and Bancroft I (2000) Comparative physical mapping of segments of the
genome of Brassica oleracea var. alboglabra that are homeologous to sequenced
regions of chromosomes 4 and 5 of Arabidopsis thaliana. Plant J 23:233-243