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Table 2 Details of the application of the method to the screening of additional candidate genes

From: A high-throughput method for the detection of homoeologous gene deletions in hexaploid wheat

gene name

sequence length (bp)1

GenBank accessions

primer sequences2 ; amplicon size1

probe sequences2

assay conditions3

samples screened; mutations detected

TaNFXL1

~630

HQ595068 - HQ595070

FNFXL1gsprb

GGCACACCAACTCCATCATG

RNFXL1gsprb

GGCTGGTTGCATCTGATATCCT

Amplicon size: ~170 bp

NFXL1prb1FAM

AACACATTCTCTTGTGACTG

NFXL1prb2VIC

AACGCACTCTCTCATGA

NFXL1prb3NED

ACATTCTCTCATTACTGACACA

FR primers: 1000 nM each

Probes: 50 nM FAM, 100 nM VIC, 100 nM NED

20 μL reaction: 18 μL stock; 2 μL DNA sample

3-step reaction: 95°C 15 s denaturation, 55°C 30 s annealing, 60°C 30 s extension - 45 cycles

2772; 14

TaCesA8

~370

HQ595065 - HQ595067

FCesA8gsprb

CCACATGGCGAAGAACACCT

RCesA8gsprb

AGTGGACGACGGTGCTGATC

Amplicon size: ~140 bp

CesA8prb1FAMCTCATACCCGCTATTGA

CesA8prb2VICCCGACCAGGTTGAG

CesA8prb3NED

ACGAGGTTGAGAACC

FR primers: 1000 nM each

Probes: 50 nM FAM, 100 nM VIC, 100 nM NED

20 μL reaction: 18 μL stock; 2 μL DNA sample

3-step reaction: 95°C 15 s denaturation, 55°C 30 s annealing, 60°C 30 s extension - 45 cycles

3762; 19

TaPLDβ1

~620

HQ595071 - HQ595073

FPLDB1gsprb

TTTGTACTTGTCTTCCTGATGTTATGC

R1PLDB1gsprb

GGCGTAGTTGGAGGCAGTCTC

R2.3PLDB1gsprb

GGTGTAGTTGGAGGCAGTCTCTC

Amplicon size: ~100 bp

PLDB1prb1FAM

TTGCTTAGAACTGCTGC

PLDB1prb2NED

CTTAGAACCGCTGCTT

PLDB1prb3VIC

TAGAACCACTGCTTC

FR primers: 1000 nM F primer, 666 nM R2.3 primer, 333 nM R1 primer

Probes: 50 nM FAM, 100 nM VIC, 150 nM NED

11 μL reaction: 9 μL stock; 2 μL DNA sample

3-step reaction: 95°C 15 s denaturation, 55°C 30 s annealing, 60°C 30 s extension - 45 cycles

3366; 11

  1. 1Approximate; some variation between homoeologues was present 2All primer and probe sequences are given in 5' - 3' directions 3All assays run in 'Standard Mode' on an ABI 7900HT (see Materials and Methods); all thermal cycling commences with 50°C 2 mins, 95°C 10 mins