Genetic diversity and molecular analysis among cotton genotypes
information of cotton microsatellite loci and selecting a
DNA Extraction
set of microsatellite primers able to differentiate between
Plants genotypes were grown in polyhouse and DNA
the various cultivars studied and to estimate the genetic
samples from the tetraploid and diploid Gossypium
distance among various cotton cultivars in Asia. Recent
species were extracted. Genomic DNA of all genotypes
studies have revealed that gene transcripts can also
was isolated at the microlevel from young leaves using
contain repeat motif, and the abundance of expressed
a CTAB-based extraction method of Altaf et al. , (1997)
sequence tags (ESTs) makes this an attractive potential
with slight modifications. Approximately 0.3 gm of fresh
source of microsatellite markers (Kantety et al 2002).
young leaf tissue was homogenized in liquid nitrogen
EST-SSRs have also been developed for Gossypium (Saha
and 0.5 ml of extraction buffer [100 mM tris-HCl (pH
et al . 2003; Han et al. 2004; Quereshi et al . 2004). More
8.0), 20 mM EDTA (pH 8.0), 1 M NaCl, 2% CTAB,
than 4lakhs Gossypium sequences were in genebank.
2% PVP-40, 1 mM 1-10, phenanthroline] and 0.2%
A total of ~70000 ESTs derived from the G.raimondii
β-mercaptoethanol in a 1.5 ml centrifuge tube with
D-genome, ~450000 ESTs from the A- genome, ~260000
the aid of a microtube pellet pestle. After incubation
from the AD tetraploid of G. hirsutm ,about ~48000
for 1 hr at 60ºC, the suspension was purified twice in
ESTs from G. arboreum , ~350 from G.herbaceum , ~300
chloroform:isoamyl alcohol (24:1) by centrifugation at
from G. barbadense , are available in the dbEST. These
10,000 rpm on a desktop micro-centrifuge for 12 min, at
EST-SSRs provide structural and functional genomic
room temperature and precipitated with an equal volume
information that will be useful for understanding cotton
of ice cold isopropanol. The recovered DNA was spooled
fiber development.
out, or pelleted by centrifuging at 10,000 rpm for 8 min,
Several EST SSR markers have been identified in
washed twice with 80% EtOH, 15 mM ammonium acetate
tetraploid cotton species and successfully used to develop
and once with 95% EtOH, air dried, and dissolved in 50
anchor SSR for cotton chromosomes to provide a basis
to 100 μl of 10mMTris buffer (pH 7.5). To this sample 2
for genetic mapping (Liu et al .,2000; Reddy et al .,2001).
μl of Rnase A (10 mg/ml) per 100 μl of dissolved DNA
There are two reports that SSR primers specific to G.
was added.
hirsutm L. have been used in mapping of G. barbadense
L.(Liu et al ., 2000a) and G. nelson L. and G. australae
Purity and Quantification test of DNA
L.(Quereshi et al 2001). EST SSR markers seems to be
Spectrophotometry was performed to determine DNA
a good tool for genomic studies and can minimize the
concentration by using Nanodrop N.D.1000 (Software
laborious cloning and screening steps of SSR development
V.3.3.0, Thermo Scientific, USA) at absorbance ratio
(Liu et al .,2000; Dayanandan et al ., 1998, 1999; Echt
260/280 nm and the quality of obtained DNA was checked
et al., 1999). Use of EST SSR markers from tetraploid
on 0.8% agarose gel. Dilution of 20 ng/µl working solution
cotton in the mapping of diploid cotton species could be
was prepared from the stock solution of the isolated
successfully utilized for marker-assisted selection (MAS)
DNAs. The 1.5 μl of DNA sample was loaded into the
and genetic diversity analysis in diploid cotton.
well of Nanodrop Spectrophotometer (Thermo Scientific,
U.S.A.) and the concentration of DNA and absorbance
Materials and Methods
at 260 nm and 280 nm were measured and the A /A
Experimental Plant Material
ratio was automatically calculated by the software. The
quality of DNA was evaluated by spectrophotometry
Gossypium species, total 24 genotypes were selected for
using the 260/280 nm absorbance ratio method and by
this comparative studies (11 G. herbaceum, 7 G. arboreum
electrophoreses on a 1% (w/v) agarose gel, and the DNA
and, 6 G. hirsutum genotypes). The genotypes of cotton
concentration estimated at 260 nm (Sambrook et al.,
were procured from Central Institute of cotton Research
1989). The stock DNA samples were stored at -20°C and
Nagpur, Regional Research Station, Anand Agricultural
working DNA samples (containing 50 ng/μL) at 4°C.
University (AAU) Anand, Main Cotton Research Station,
Surat, Regional Cotton Research Station Viramgam and
Regional Cotton Research Station, Bharuch.