Biology Reference
In-Depth Information
Diana in eight SNPs and a single nucleotide dele-
tion. The HRM assay that differentiates between
alleles associated with resistance and suscepti-
bility is based on the 397bp-long region encom-
passing seven of the SNPs (Figure 14.4).
PCR amplification was performed in a 10 μl
reaction volume containing 10 ng of genomic
DNA as a template, 1
always associated with susceptibility to the dis-
ease, while three haplotypes are associated with
disease resistance. The three resistant haplotypes
were termed R1 (from cv. Salinas), R2 (from
accession PI 491224), and R3 (from
L. serriola
accession UC96US23).
PCR amplification was performed according
to published protocol (Simko et al. 2009). A 10
μl reaction volume contained 10 ng of genomic
DNA as a template, 1
PCR buffer, 0.75 U
Ta q
polymerase, 0.125 mM of each dNTP (all
from New England Biolabs, Ipswich, MA), 1
×
×
LCGreen Plus Melting Dye (Idaho Technology,
Salt Lake City, UT), and 0.25 μM of each primer
(AY207423b-F: 5'- TCC TGA ATT CCC AAA
ATA CCC -3', and AY207423b-R: 5'- TAG CCC
ATT TAA TGC CAT GC -3'). The reaction
was filled to the final volume with nuclease-free
water; 15 μl of mineral oil was added to prevent
evaporation during HRM analysis. PCR was per-
formed with an initial denaturation of 95
◦
Cfor
2 min, followed by 45 cycles of 95
◦
C for 30 s,
66
◦
C for 30 s, and 72
◦
C for 30 s, with final
extension of 72
◦
C for 5 min. To facilitate het-
eroduplex formation, samples were heated to 95
◦
C for 30 s, followed by cooling to 25
◦
C for 30s
(this step can be omitted if only homoduplexes
are analyzed). Figure 14.3 (top row) shows dif-
ferent melting profiles of the two alleles.
PCR buffer, 1.5 mM
MgCl
2
,0.6U
Ta q
polymerase, 0.2 mM of each
dNTP (all from New England Biolabs, Ipswich,
MA), 1
×
LCGreen Plus Melting Dye (Idaho
Technology, Salt Lake City, UT), and 0.25 μM
of each primer (Cntg10192-F: 5'- CTC GTT
TTC AAC ACC GAC AA -3', and Cntg10192-
R: 5'- TAG GTG GGT CCG ACT TTG AG -3').
The reaction was filled to the final volume with
nuclease-free water; 15 μl of mineral oil was
added to prevent evaporation during HRM anal-
ysis. PCR was performed with an initial denatu-
ration of 95
◦
C for 2 min, followed by 45 cycles
of 95
◦
C for 30 s, 61
◦
C for 30 s, and 72
◦
Cfor
30 s, with final extension of 72
◦
Cfor5min.To
facilitate heteroduplex formation, samples were
heated to 95
◦
C for 30 s, followed by cooling to
25
◦
C for 30s (this step can be omitted if only
homoduplexes are analyzed). Different melting
profiles of the four haplotypes are shown in Fig-
ure 14.3 (middle row).
×
Lettuce Dieback
The
Cntg10192
marker locus on LG 2 is linked to
the
Tvr1
gene that provides complete resistance
against lettuce dieback (Simko et al. 2009; Simko
et al. 2010a; Simko et al. 2010b). This locus was
sequenced from 73 accessions of four
Lactuca
species (
L. sativa
,
L. serriola
,
L. saligna
, and
L.
virosa
) and the sequences were deposited into
the NCBI database under numbers GQ341366
to GQ341438. The 349bp-long fragment con-
tains 11 SNPs that differentiate six haplotypes
(Simko et al. 2010b). The HRM assay is based
on the 185bp-long fragment that encompasses
three of the SNPs and can be used to distin-
guish four haplotypes identified in
L. sativa
and
L. serriola
(Simko et al. 2009, Simko et al.
2010b) (Figure 14.4). One of the haplotypes is
Lettuce Mosaic
The HRM assay was developed to distinguish
two resistant (
mo-1
1
and
mo-1
2
) and one suscep-
tible (
Mo-1
) allele of the
mo-1
resistance gene
located on LG 4 (Nicaise et al. 2003). The resis-
tant alleles were first identified in cv. Gallega
de Invierno (von der Pahlen and Crnko 1965)
and accession PI 251245 (Ryder 1970), respec-
tively. The full-length sequence from susceptible
cv. Salinas (NCBI database number: AF530162)
is 1,032bp long (Nicaise et al. 2003). The
mo-1
2
allele differs from
Mo-1
in two SNPs,
while
mo-1
1
differs from
Mo-1
in two differ-
ent SNPs and one 6bp-long deletion. The HRM
Search WWH ::
Custom Search