The wheat molecular markers Ta1-FFT, Ta6-SFT and TaWIVRV assayed on Roche LightCycler 480 using High-Resolution Melting analysis
Marker
Primer sequence 5’-----3’
Length of
SNP alleles
Tm
MgCl2
Pre-melt
Post-melt
Temperature
(NCBI sequence)
(forward/reverse)
fragment
(Berkut/Krichauff)
(oC)
(mM)
slider (oC)
slider (oC)
shift (oC)
Ta1-FFT
AGGCATCCTTCCGCATCAAC
212 bp
TT/TC, TC/CC
60
2.0
82-84
93-94
1
(AB088409.1)
ATCGTGGCAGAAGTGCGTTC
Ta6-SFT
AATGAAGCTCTTGCATCTCATCAGT
203 bp
G/A, T/C, A/G,
65
3.0
76-77
84-85
5
(AB029887.1)
CCACCCAAACTATATCGATTCTACG
TaWIVRV
TGCTCGACGGTGAGGATCTAT
62
3.0
87-88
92-93
5
(AF069309.1)
GGGCCAAGAGCTTGATTAATTG
-----/CAGTA
275 bp
G/C, T/C, T/C,
G/A, G/A, T/C
The wheat molecular markers Ta1-FFT, Ta6-SFT and TaWIVRV (from left to right): (A)
Fragments amplified from Berkut, Krichauff and nulli-tetra lines of Chinese Spring N7AT7B,
N7BT7A, N7DT7A; (B) High-resolution melting profiles for gene amplicons from Berkut (in
red), Krichauff (in green), Berkut/Krichauff doubled haploid lines. Berkut alleles are in red,
Krichauff alleles in blue and Berkut/Krichauff heterozygote alleles are in green. The upper
chart shows the normalized temperature-shifted melting curves, and the lower chart shows
the difference curves using Berkut’s melt profiles as the baselines.
1000
300
200
100
Relative signal difference
Relative signal (%)
B
Temperature (oC)
N7DT7A
N7BT7A
N7AT7B
Krichauff
Berkut
N7DT7A
Ta.WIVRV
N7BT7A
N7AT7B
Krichauff
Berkut
N7DT7A
Ta6-SFT
N7BT7A
N7AT7B
Berkut
2000
Krichauff
Ta1-FFT
A
The wheat molecular markers Ta1-FFT, Ta6-SFT and TaWIVRV (from left to right): Highresolution melting profiles for gene amplicons from wheat varieties from diverse origins.
The upper chart shows the normalized temperature-shifted melting curves, and the lower
chart shows the difference curves using Berkut’s melt profiles as the baselines. Lines with
Berkut-like alleles are in red. Lines with Krichauff-like alleles are in blue. Lines with rare
alleles are in other colours.
Ta6-SFT
Relative signal difference
Relative signal (%)
Ta1-FFT
Temperature (oC)
Ta.WIVRV
The wheat molecular marker Ta1-SST: (A) Fragments amplified from Berkut, Krichauff,
Berkut/Krichauff heterozygote, Berkut/Krichauff doubled haploid lines, Chinese Spring and
group-7 nulli-tetra lines, and (B) Sequence structure of fragments amplified from Berkut and
Krichauff and intron-exon structure of the wheat 1-SST gene. Exons are indicated by filled
Berkut/Krichauff doubled haploid lines
N7AT7D
Berkut
Krichauff
Heterozygote
A
Chinese Spring
N7AT7B
N7BT7A
boxes and introns by solid lines. Numbers represent lengths of exons and introns.
2000
2.0 kb
1.8 kb
1800
1.6 kb
1600
B
1400
271
1231
Berkut
(1843 bp)
Stowaway-like
insertion
341
156
271
1041
341
Krichauff
(1653 bp)
406
Wheat 1-SST gene
(3326 bp)
161 146
872
1030
702
9
Primer AB029888-15F
Primer AB029888-19R
(1304-23 bp)
(2963-82 bp)
BATAVIA
BEYROUTH 1
PELADA
SALAMANCA 10
LA PREVISION
CLUBHEAD
DALMATIA 2
SAPPORO
AMC 61
WYLAH
Chara
Samnyt16
Kukri
Sokoll
Westonia
Samnyt16
RAC875-2
Tasman
Excalibur
Krichauff
Cranbrook
YITPI
W63
ELLISON
AKADARUMA
SALONICA 17
ALTAR
W43A
OLYMPIC
SMYRNA 13
GLUYAS EARLY
IRAN 158
KEFENG
BATAVIA
NW19A
KATYIL
MOROCCO 21
AMC 106
SAPPORO
DALMATIA 2
CLUBHEAD
LA PREVISION
SALAMANCA 10
W58
W145
W49A
INDIA 259
PUNJAB 7
ASURE BUGDAY
W75
REES
SION
TUNIS 24
H 501
ROGUE IN BLE DU OASI
YANGMAI 3
GABO
T. AESTIVUM SSP SPAE
NW96A
AMC 512
KANDAHARY
W45B
WARREN
KOPARA 73
H 1287
SATURNO
GIZA 139
H 1160
AMC 125
H 865
ARNHEM
MOLDOVA
D.E.S. 0043
HERMITAGE
KARIZIK
SURHAK JUBILEJNYJ
BOWERBIRD
QAL 2000
NW99A
AMC 83
VERANOPOLIS
BANKS
BELADI 42
FRONTANA
BOUNTY
DRYSDALE
MOROCCO 59
KIRAN
INSIGNIA
SEVILLE 22
W216
W143
INDIA 211 (?)
W139A
H45
ALLORA
RELIN
PERSIA 80
VARNA 6
AMC 136
BEYROUTH 1
PELADA
AMC 53
MOROCCO 16
RUFUS
AMC 61
BANKS
LERMA ROJO
PORTUGAL 102
1.0
PUMAFEN
1.2
WYLAH
kb
2.0
1.8
1.6
1.4
Berkut
The wheat molecular marker Ta1-SST: Fragments amplified from wheat varieties from
diverse origins (more details in Supplemental file 1).