Molecular Plant Breeding 2016, Vol.7, No.14, 1
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10
3
Figure 1 GISH of
mitotic metaphase chromosomes of two cultivated allotetraploid cotton species
G.hirsutum
(AD)
1
and
G.barbadense
(AD)
2
with all 3 A diploid cotton gDNA probes respectively (all probes were labeled with digoxigenin, and red
fluorescence signals were observed on A sub-genome chromosomes in each image). Fig.1-a To 1-c: GISH of
G.hirsutum
var. zhong
16
with diploid A genome species
G.herbaceum var. hongxing
(A
1
),
G.arboreum var. shixiya 1
(A
2
),
G.herbaceum wild species
arfrium
(A
1-a
) gDNA probe rsepectively, blocking with ssDNA (In Fig.1-c, six arrows showed GISH-NORs, of which two green
arrows denote the GISH-NORs in A sub-genome chromosomes and other four white arrows denote the GISH-NORs in D
sub-genome chromosomes of (AD)
1
). Fig.1-d To 1-f: GISH of
G.barbadense
var. Xinhai 7
also with diploid A genome species A
1
,
A
2
,
and A
1-a
gDNA probe rsepectively, blocking with ssDNA (In Fig.1-f, six arrows also showed the signals of GISH-NOR, of which
two green arrows denote the GISH-NORs in A sub-genome chromosomes and four white arrows denote in D sub-genome).
Bars=10
m
Table 1 Distribution of GISH-NORs in (AD)
1
and (AD)
2
by GISH with all A and D diploid gDNA probes
Target
gDNA Probe
Chromosome
Pairs
Size
Intensity
Position
(AD)
1
A
1-a
A sub-genome
1
Major
weak
terminal
D sub-genome
2
Major
weak
terminal
A
1
,A
2
A sub-genome
0
-
-
-
D sub-genome
0
-
-
-
all 13 diploid D
genome species
A sub-genome
1
Major
strong
terminal
D sub-genome
2
Major
strong
terminal
(AD)
2
A
1-a
A sub-genome
1
Major
terminal
D sub-genome
2
Major
terminal
A
1
, A
2
A sub-genome
0
-
-
-
D sub-genome
0
-
-
-
all 13 diploid D
genome species
A sub-genome
1
Major
strong
terminal
D sub-genome
2
Major
strong
terminal
1.2 GISH of (AD)
1
and (AD)
2
with all 13 diploid D cotton gDNA as probes respectively
Except the D
6
(
G.gossypiodies
) gDNA probe, GISH of the somatic metaphase chromosomes of (AD)
1
and
(AD)
2
both with other 12 diploid D gDNA as probe respectively, blocking with ssDNA (As shown in Figure 2 and 3), the
red fluorescence signals were observed on the short D sub-genome chromosomes of (AD)
1
and (AD)
2
. In addition,
three pairs of major GISH-NORs were detected, of which one in the longer A sub-genome chromosomes (green
arrows showed in each image), and other two in the D sub-genome chromosomes (white arrows in each
image),and the specific distribution about GISH-NORs were shown in Table 1. However, there were some
differences on the fluorescence signal strength generated by each D genome species probe. GISH of (AD)
1
and
(AD)
2
with D
6
gDNA probe respectively, the red fluorescence signals were not only distribute on the D
sub-genome chromosomes but also on A sub-genomes (As shown in Figure 2-h and Figure 3-h), and we could not
distinguish the A and D sub-genome of (AD)
1
and (AD)
2
, and three pairs of major GISH-NORs were also
observed. This showed a great difference between the D
6
genome and the other 12 D genomes, and the result
illustrated that diploid D
6
genome may contain plenty of A genome repeat sequences, it is a very special genome
species in the diploid D genomes.
1.3 DA value analysis based on GISH of (AD)
1
and
(AD)
2
with diploid A, D genome species probes
DA (distinguishing ability) value reflect the genetic relationship between the diploid A or D genomes and the
tetraploid genomes (Markova et al., 2007). DA values (As shown in Table 2) generated by A
1
, A
2
and A
1-a
gDNA
probes to (AD)
1
was 0.361, 0.358 and 0.369 respectively. It showed the A
1-a
gDNA possess the strongest ability to
recognize the A sub-genome chromosomes of (AD)
1
, while the A
1
and A
2
gDNA possess same ability to
recognize the A sub-genome chromosomes of (AD)
1
, that’s to say the A
1-a
genome had closer genetic relationship
with the A sub-genome of (AD)
1
. And DA values generated by A
1
, A
2
and A
1-a
gDNA probes to (AD)
2
were 0.343,
0.346 and 0.352 respectively. the results also showed the A
1-a
gDNA possess the strongest ability to recognize the
A sub-genome chromosomes of (AD)
2,
therefore, the A
1-a
genome also had closer genetic relationship with the A
