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RESULTS of six seedlings7 (Fig. 1, yellow bars). Additionally, we used a new

Genome sequencing and assembly
We selected a fourth-generation inbred line of the F. vesca ssp.
vesca accession Hawaii 4 known as H44 for sequencing primarily
because of its amenability to high-throughput genetic transforma-
tion. The Hawaii 4 accession was used for transfer DNA (T-DNA)
insertional mutagenesis5,6, as well as transposon and activation
tagging. H4x4 is day neutral, sets abundant seeds on self-pollination
and completes a life cycle in 46 months regardless of season. It has
white-yellow fruit and produces new plants from modified stems
called stolons.
We used the Roche/454, Illumina/Solexa and Life Technologies/
SOLiD platforms to generate 39 combined average coverage (Online
Methods). A summary of the input sequence data used for the assem-
bly is presented in Supplementary Table 1. Over 3,200 scaffolds were
assembled with an N50 of 1.3 Mb (Supplementary Table 2). Over
95% (209.8 Mb) of the total sequence is represented in 272 scaffolds.
Resequencing using Illumina confirmed the high quality of the assem
bly, with 99.8% of the scaffolds and 99.98% of the bases in the assem-
bly being validated by perfect-match Illumina reads with an average
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method to identify segregating SNP markers through direct Illumina
sequencing of a reduced complexity AluI digestion of the bin set seed-
ling DNA for anchoring 70 additional scaffolds of over 100,000 kb in
length to the genetic map. Three scaffolds mapped to two locations
on the genetic map and were split into two at regions of low coverage.
Thus, a total of 204 genome sequence scaffolds (including the three
split scaffolds) containing 198.1 Mb of sequence data (~94% of the
total scaffold sequence) was anchored to the FV FN map using 390
markers. We assembled the scaffolds into seven pseudochromosomes,
numbered according to the linkage group nomenclature used in a
previous study8.
Although a comprehensive molecular karyotype has yet to be estab-
lished for F. vesca, researchers in a previous study9 identified, by fluo-
rescent in situ hybridization (FISH), three pairs of 45S (18S-5.8S-25S)
loci and one pair of 5S loci that co-localized with one of the pairs of 45S
loci in an unspecified accession of F. vesca. We also found these karyo-
typic features in F. vesca H4x4 and identified tentative correspondences
between two cytologically marked chromosomes and genomically
defined pseudochromosomes using mitotic (root tip) chromosomes

depth of approximately 26 (Supplementary Fig. 1). The F. vesca
H4x4 genome size was estimated at ~240 Mb using flow cytometry,
with Arabidopsis thaliana (~147 Mb) and Brachypodium distachyon
(300 Mb) serving as internal controls (Supplementary Table 3).
Anchoring genome sequence to the genetic map
We aligned and oriented the scaffolds of the assembly to the diploid
Fragaria reference linkage map (FV FN) and its associated bin map7
(Fig. 1). Of 272 F. vesca H4x4 sequence scaffolds that were com-
posed of over 10,000 bp (a total of 209.8 Mb of scaffold sequences and
embedded gaps), 131 were anchored to the FV FN map. The scaffolds
were anchored by 320 genetic markers, including 234 mapped in the
full FV FN progeny6 (Fig. 1, blue bars) and 86 mapped in a bin set
hybridized to 25S (red) and 5S (green) rDNA probes (Online Methods
and Supplementary Fig. 2). Chromosome G displayed a strong distal
25S signal and a proximal 5S signal, whereas chromosome F displayed
a strong distal 25S signal and chromosome D displayed a weak distal
25S signal. The 5S probe sequence had sequence homology to two
small scaffolds that are not mapped to pseudochromosomes and one
scaffold that maps to pseudochromosome VII at the locus defined by
marker EMFv190. The 25S sequence had 32 matches of >90% sequence
identity, of which 30 were unmapped scaffolds of less than 1.7 kb.
Pseudochromosome VII, with mapped scaffolds containing 25S and
5S sequences at distal and proximal locations, respectively, appears
to correspond to chromosome G. Pseudochromosome VI, which
also contains 25S sequences in a mapped scaffold may correspond

Marker cM LG 1 Kb Marker cM LG 2 Kb Marker cM LG 3 Kb Marker cM LG 4 Kb Marker cM LG 5 Kb Marker cM LG 6 Kb Marker cM LG 7 Kb

EMFv016 (0.0)
VT-846 (0.0) 589 VT-008A (0.0) 99 1,497 Chr4 (0.0) AC49 (0.0) VT-704 (0.0) Chr7 (0.0) 769
359 EMFv029 (2.0) 902
114 1,929
1,445 503 355 808 AC31 (3.0) 1,016
639 343 3,447
232 203
EMFvi072 (6.3) VT-837 (8.0) 193 UFFxa02H04 (8.0) 1,854 EMFvi108 (7.0)
1,875 237 2,874 1,386
EMFvi099 (10.0) 216
542 RC1173,VI:07 (10.0) 337
495 GP.V:21 (10.0) 1,074 361
779 EMFvi025 (12.0) 5,161
RC0052 (14.0) 1,193 FPS.III:13 (13.0) 91 PC14 (12.0) 388 419
EMFn049 (12.8) 885 EMFn235 (16.0) VT-398 (15.0) 2,261 681 1,392 CHS (13.0)
1,079 1,443 EMFn228 (15.0)
EPpCU9910 (14.6) 500 EMFn121 (17.0) 881 EMFv021 (14.0)
ADH (18.0) EPpCU1830 (17.0)
EMFn148 (20.0) 186 168 EMFv190 (16.0)
923 VT-734 (18.0) 159
APB (22.0) 1,898 833 115 EPpCU9257 (18.0)
VT-673 (21.0) 584 CEL2 (20.0)
3,435 1,594
341 AC24 (20.0) 1,111 1,811 4,105
VT-714 (26.0) VT-744 (24.0) 691 EMFn213 (21.0)
UDF002 (22.2) 461 AG53 (22.0)
2,682 1,027 995
UDF017 (27.0) 132 EMFn110 (26.0) CFVCT019 (24.0)
Fvi11 (31.0) UDF016 (28.5) 1,398 EFvVB2179,IV:26 (26.0)
EMFn136 (26.0) 1,992 2,549 1,497
CFACT104.V:29 (29.0) 1,017
EMFn214 (34.0) 694 CFVCT032 (31.0)
545 LOX (28.0) 2,544 EMFn185 (32.0)
424 UDF004 (33.0) 1,178 1,040
324 920 EMFv180 (30.0) 495 EMFvi008 (30.0)
EMFn134 (38.0) EMFn034 (35.0) 1,419 EMFn123 (35.0) 486
626 EMFn111 (32.0)
UDF006 (35.0) 1,278 546
1,900
2,767 2,597
EMFn170 (39.0) VT-756 (35.0) CFVCT024 (37.0) 796 1,226
UFFxa15H09 (44.0) 2,262 EPpCU9223 (36.0)
EMFn125 (41.0) 2,850 UDF009 (39.0) EMFvi133 (41.0)
CFACT099,I:41 (37.0) 1,238 CFVCT036 (43.0)
RC0537,II:47 (47.0) EMFvi038 (43.0) 1,772
709 875 1,264 EMFv006 (44.0)
681 VT-816 (40.0) EMFv104 (45.0)
363
PES,I:21 (41.1) 594 UWC513169,II:50 (50.0) 110 857 404 374 3,552 VT-838 (41.0)
677 246 AG33 (45.0) VT-674 (48.0) 1,416
550 EMFn207 (48.0) 1,378
713 EMFxa379796 (55.0) CFVCT017 (50.0)
940 EMFxa381788 (50.0) 1,461
813 EFvVB1231.IV:46 (46.0) EMFvi018 (49.0)
153 535 4,676 (47.0)
VT-725 (53.0) 376
F3H (47.5) 106 DFR (59.0) EMFn162 (51.5) 2,447 2,607
406 839 2,458
75 1,790 EMFn238 (53.0) ZIP (56.0)
EMFn182 (51.0) 111 EMFv003 (63.0) ACO (58.0)
181 2,088 EMFn010 (55.5)
468 CFVCT021 (65.0) 1,052 EMFvi136 (53.0) 1,481 EMFn153 (60.0)
512991CT47 (54.0) 80
283 111 998 2,335
74 83 141 3,270
136 647 EMFv023 (56.0)
EMFn128 (56.5) 895
480
EMFv183 (72.0) 2,778 377 978 2,078 903
794 VT-659 (59.0)
720 VT-861 (60.0) RC1247 (68.0)
578 CFVCT012 (68.0) 100 UWC512934.VI:70 (70.0)
183 1,669 686
336 2,869
900 VT-076 (73.0)
231 632
EMFv164 (65.6) 150 595 511 QR (75.0) Omt1 (66.0)
468 264 182
457 385 UWC513004.IV:68 (68.0) EFvVB2013.V:73 (73.0) 67 898
VT-679 (78.0)
255 858
237 1,272 653
1,000 570 1,174
244
UWC513099,I:90 338 709 420
(74.0) 350 CEL1 (75.0) 439
406 399
420 2,460
465 UWC512940.VI:90 (90.0)
539 UDF008 (80.0) MEX.VII:90 (80.0)
208
258
247
3,083 599
VT-753 (86.0) 506
306
1,469
RC1635,I:90 (90.0)
138
177
206
479
1,327
156

Figure 1 Anchoring the F. vesca genome to the diploid Fragaria reference map, FV FN. Scaffolds representing 198.1 Mb of scaffolded sequence with
embedded gaps (99.2% of all contiguous sequence over 10 kb in length) were anchored to the genetic map with 390 genetic markers. Blue scaffolds
were anchored and oriented using map positions of markers in the full FV FN progeny, whereas the yellow scaffolds were anchored to mapping bins.

110 VOLUME 43 | NUMBER 2 | FEBRUARY 2011 Nature Genetics