SUPPLEMENTARY INFORMATION (Veerappan et al_Planta)
SUPPLEMENTARY RESULTS
Map-based cloning of gce1 mutation
For mapping the elevated luminescence phenotype in gce1mutant, we used a map-based cloning
strategy. A total of 50 individual gce1 mutant plants were identified from F2 segregating
population from the cross gce1 x Ler (Landberg erecta). Initial scoring of recombination events
using 20 SSLP markers spanning all five Arabidopsis chromosomes localized gce1 mutation to
the middle of Arabidopsis chromosome 2 between markers CER451559 and CER460534 9 (Fig.
2a). Further genotyping using 11 additional SSLP markers located the gce1 mutation within a
418 Kb region between CER449629 and CER458055. For high resolution mapping of the gce1
locus, 1488 homozygous gce1 mutant plants were selected from a segregating F2 mapping
population. Individual DNA samples were genotyped for recombination by using two robust
SSLP markers CER449629 and CER458055. Out of 1488 plants scored, 54 plants showed
recombination for CER449629 and 12 plants for CER458055 (Fig. 2a). Further genotyping of
recombinant plants using additional CAPS and SSLP markers narrowed the gce1 mutation to a
genomic region between BAC clones T6B20 and T9D9 within a 71 Kb mapping interval which
includes 21 open reading frames (Fig. 2a). Candidate genes within the mapping interval were
sequenced.
Map-based identification of the KanR transgene locus
Despite multiple rounds of TAIL-PCR reactions, we were unable to obtain any novel genomic
sequences flanking the GSTF8::LUC T-DNA of the KanR locus on chromosome IV. The
identification of the genomic location of T-DNA border sequences using TAIL-PCR is often
difficult because of the complex nature of T-DNA insertions and multiple copies in the same
locus (O'Malley et al., 2007; Thole et al., 2009). The presence of two independent GSTF8::LUC
T-DNA loci in hsi2-4 mutant may also produce false-positives due to the random PCR
amplifications during TAIL-PCR. Hence, to identify the exact location of the KanR transgene
locus, we took a genetic approach. Our preliminary mapping showed that the KanR locus is
linked to the right arm of chromosome IV (Supplementary Fig. S2a). Further fine mapping using
several additional new SSLP markers narrowed down the chromosome IV locus to 800 Kb
mapping interval (Supplementary Fig. S2b). To identify more recombinants within the mapping
interval, we screened ~ 25,000 F2 seedlings by luminescence imaging and selected 1250
seedlings with “no luminescence” phenotype from the mapping population. Two robust SSLP
markers CER466357 and CER451652 were used to score the recombination events in 1250 F2
plants. Additional SSLP and CAPS markers placed the KanR locus within 255 Kb mapping
interval between a CAPS marker CER447377 (DraII) and an SSLP marker CER450334
(Supplementary Fig. S2b).
To pinpoint the exact location of the KanR transgene locus, we used differential amplification of
long PCR fragments between Col-0 wild-type and hsi2-4 mutant. We designed 29 overlapping
long PCR primer pairs covering the entire 255 Kb mapping interval to amplify ~ 10 Kb
fragments using Col-0 and hsi2-4 genomic DNA (Supplementary Fig. S2b). In all the genomic
locations, we can expect to see specific long PCR amplification products in both Col-0 and hsi24 mutant but near the vicinity of T-DNA insertion, long PCR primers can amplify only in Col-0;
whereas in hsi2-4 mutant there will be no amplification because the amplification size is larger
than the designed 10 Kb due to the T-DNA insertion. Long-PCR amplifications using the
genomic DNA from Col-0 and hsi2-4 mutant indicated that one of the primer P12 (9.8 Kb
fragment lies between 11513301 bp -11523001 bp on chromosome IV) shows amplification only
in Col-0 whereas in the hsi2-4 mutant there was no amplification detected. We have designed
four overlapping primers of ~4 Kb size to cover the entire 10 Kb genomic region of primer P12
and one of the primers P12-G showed amplification only in Col-0 but failed to amplify in the
reporter line. To identify the exact location of the T-DNA insertion, PCR products obtained
using the genomic primer P12-G-F and T-DNA left border primer pBI101-LB2-R were
sequenced to find the genomic sequences flanking the KanR locus transgene. The KanR locus TDNA insertion was located in the intergenic region between 11521889 bp -11521890 bp on
chromosome IV.
SUPPLEMENTARY MATERIALS AND METHODS
Genetic crosses and genotyping
To study the inheritance of hsi2-4 mutation, hsi2-4 mutant was backcrossed to the WTluc parent.
Pollen from the WTluc plants was used to pollinate emasculated gce1 flowers. Successful crosses
were identified by luciferase imaging of F1 seedlings. F1 plants were allowed to self and
luciferase imaging was performed using F2 seedlings segregating for hsi2-4 phenotype.
Backcrosses were repeated four times to remove extraneous mutations caused by EMS in the
hsi2-4 background. Genetic complementation was performed by crossing hsi2-4 mutant (male) to
hsi2-2 (female). F1 seedlings from successful crosses were used for luciferase imaging. To
introgress the GSTF8::LUC transgene into hsi2-2 T-DNA insertion background, plants
homozygous for T-DNA insertion in HSI2 were emasculated and pollinated using pollen obtained
from WT
luc
plants carrying GSTF8::LUC transgenes. Successful crosses were confirmed by
luciferase imaging in the F1 generation and F1 plants were allowed to self. Seedlings showing
luminescence were selected in the F2 generation and individual plants homozygous for the TDNA insertion in HSI2 were identified by PCR genotyping. Plants homozygous for hsi2-2 TDNA insertion and GSTF8::LUC transgene were also confirmed in the F3 generation based on
PCR genotyping and segregation for the luminescence phenotype respectively.
For the removal of the GSTF8::LUC transgene from the hsi2-4 mutant background, hsi2-4
mutant (male) was out crossed to Col-0 (female). Successful crosses were confirmed by
luminescence imaging in the F1 generation and plants were allowed to self-pollinate. Individual
plants showing “no luminescence” were selected in the F2 segregating population and genotyped
using the CAPS marker hsi2-4-BsmI to identify the plants homozygous for the hsi2-4 point
mutation. For co-segregation analysis of hsi2-4 mutation and GSTF8::LUC transgenes,
individual F2 seedlings from the cross Col-0 x hsi2-4 were used for luminescence imaging and
hsi2-4-BsmI CAPS genotyping. To create the double mutants of hsi2-2 x hsl1 and hsi2-4x hsl1,
crosses were performed. Successful crosses were confirmed in F1 generation by PCR genotyping
and allowed for self-pollination. Homozygous double mutants were identified from F2
segregating population based on genotyping using PCR markers and CAPS marker hsi2-4-BsmI.
Homozygous hsi2-2 and hsl1 T-DNA insertions were identified by PCR genotyping using a
combination of gene specific (Right-RP and Left-LP) and T-DNA border specific (LBb1.3)
primers (Supplemental Table 8). For genotyping the hsi2-4mutation, a CAPS marker was
developed based on the point mutation in the HSI2 gene. hsi2-4-BsmI PCR primers
(Supplemental Table 8) were used to amplify 2.2 Kb PCR fragment from the genomic DNA and
digested with BsmI restriction endonuclease. Restriction digestion products were separated and
analyzed on agarose gel to find the plants homozygous for hsi2-4 mutation.
Genetic mapping and TAIL-PCR
Mapping population was developed by outcrossing the hsi2-4 mutant in Col-0 background
(male) with an alternative ecotype Ler (female). Successful crosses were identified by luciferase
imaging of F1 seedlings. To map the gce1 recessive mutation, seedlings showing constitutively
elevated luminescence phenotype were identified from F2 segregating populations. Crude
genomic DNA was extracted from two to three week old gce1 mutant plants and genotyped for
recombination frequency using SSLP and CAPS markers developed using the CEREON
database (Jander et al., 2002). To map the chromosomal locations of GSTF8::LUC, we used the
same hsi2-4 x Ler mapping population that we used for map-based cloning of hsi2-4 mutation.
TAIL-PCR conditions and details of the arbitrary degenerate (AD) primers were described before
(Liu et al., 1995; Sessions et al., 2002). Briefly, pBI101 T-DNA border specific primers including
pBI101-LB-1R, pBI101-LB-2R and pBI101-LB-3R were used in combination with a pool of
four different AD primers AD1, AD2, AD3 and AD6 in subsequent TAIL-PCR reactions. PCR
products were separated on agarose gel, specific DNA bands were gel purified using QIAquick
gel extraction kit (Qiagen) and subcloned into pGEM-T-easy vector (Promega) for sequencing.
For PCR genotyping of the KanS locus, pBI101 left border specific primer LBb1.3 were used in
combination with genomic primers KanS-LP and KanS-RP. For PCR genotyping of the KanR
locus, T-DNA left border primer pBI101-LB-L2 was nested with genomic primers KanR and
KanR. All the PCR primers used in TAIL-PCR, CAPS and SSLP based genotyping are listed in
Supplemental Tables S8-S10.
SUPPLEMENTARY REFERENCES
Jander G, Norris SR, Rounsley SD, Bush DF, Levin IM, Last RL (2002) Arabidopsis map-based
cloning in the post-genome era. Plant Physiol 129: 440-450
Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of
Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J
8: 457-463
O'Malley RC, Alonso JM, Kim CJ, Leisse TJ, Ecker JR (2007) An adapter ligation-mediated
PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome.
Nature Protocols 2: 2910-2917
Sessions A, Burke E, Presting G, Aux G, McElver J, Patton D, Dietrich B, Ho P, Bacwaden J,
Ko C, Clarke JD, Cotton D, Bullis D, Snell J, Miguel T, Hutchison D, Kimmerly B, Mitzel
T, Katagiri F, Glazebrook J, Law M, Goff SA (2002) A high-throughput Arabidopsis reverse
genetics system. Plant Cell 14: 2985-2994
Thole V, Alves SC, Worland B, Bevan MW, Vain P (2009) A protocol for efficiently retrieving
and characterizing flanking sequence tags (FSTs) in Brachypodium distachyon T-DNA
insertional mutants. Nature Protocols 4: 650-661