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

260

280

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.

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