Molecular Plant Breeding 2016, Vol.7, No.14, 1
-
10
8
3 Materials and Methods
We used allotetraploid cultivated cotton species (2n = 4x = 52) (AD)
1
(
G. hirsutum
var. Zhongmiansuo 16
) and
(AD)
2
(
G. barbadense
var. Xinhai 7
) as target chromosomes. The Probes were made from diploid cotton (2n = 2x
= 26) genomic DNA (gDNA), including all 3 A genome species
G. herbaceum var. hongxing
(A
1
), and
G.
arboreum var. shixiya 1
(A
2
), and
G. herbaceum
wild
var. arfrium
(A
1-a
); and all 13 D genome species, including
G.
thurberi
(D
1
),
G. armourianum
(D
2-1
),
G. harknessii
(D
2-2
),
G. davidsonii
(D
3-d
),
G. klotzschianum
(D
3-k
),
G. aridum
(D
4
),
G. raimondii
(D
5
),
G. gossypiodies
(D
6
),
G. lobatum
(D
7
),
G. trilobum
(D
8
),
G. laxum
(D
9
),
G. turneri
(D
10
)
and
G. schwendimanii
(D
11
). All of the plant materials are being taken from National Wild Cotton Germplasm
Nursery in Sanya city, Hainan, China.
Total gDNA was extracted and purificated from immature leaves using the CTAB method (Song et al., 1999)
.
The
purified total gDNA was cut off with 120
℃
10 min, and examined with 0.8% agarose gel electrophoresis for their
fragment size, which was generally appropriate in 300-600bp, and then tagged with markers. The gDNA probes
were labeled with DIG-High-Prime labeling system (Roche Company, Germany), according to the standard
operating procedures. The preparation of mitotic metaphase chromosomes which derived from the root tip cells
and the procedure of GISH referenced to the method of Wang (Wang et al., 1999).
The hybridization signals were observed using a fluorescence microscope (Ziess Axioskop 2 plus). Images were
captured by ISIS (in situ imaging system) software by adjusting their brightness and contrast. And also used this
software to calculate the DA (distinguishing ability) value which was described by Markova et al (2007). And
used Adobe Photoshop 7.0 software makes the plate.
Acknowledgment
This work was supported by the Exploring and Ultilization of Economic Crop Germplasm Resources(2013BAD01B03), the National
Agriculture Research System of Cotton (CARS-18-36) and the Subproject of the Major Projects of Genetically Modified Organisms
Breeding (2011ZX08005002-009) and also the Science and Technology Plan of the Jiangxi Province (20141BBF60011).
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