International Journal of Horticulture 2015, Vol.5, No.12, 1
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10
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Table 1 Names, source, origin and type of all squash genotypes used in the study
Genotype
Source
Origin
Type
PI 506466
NPGS
, USA
Russian Federation
Squash
1.1
PI 292014
NPGS
, USA
Israel
Squash
PI 518688
NPGS
, USA
United States
Acorn Squash
PI 506467
NPGS
, USA
Ukraine
Squash
PI 615119
NPGS
, USA
United States
Spaghetti Squash
PI 136448
NPGS
, USA
China
Squash
PI 518687
NPGS
, USA
United States
Acorn Squash
PI 216032
NPGS
, USA
India
Squash
Matrouhy
Local Landrace
Egypt
Squash
Butternut
Bentley Seeds Inc., USA
United States
Winter Squash*
Yellow Crookneck
Bentley Seeds Inc., USA
United States
Squash
Shamamy
Local Landrace
Egypt
Squash
Copi
Local Landrace
Egypt
Squash
Eskandrani
Horticultural Research Institute
Egypt
Squash
*
Cucurbita moschata
out in spring-summer season of 2014. The soil of the
experimental site was sandy soil (85.21% sand, 11.5%
silt and 3.29% clay) with pH 8.10 and EC 0.87 dsm
-1
.
Before planting, the experimental location was prepared
three months before transplanting. During preparation,
a rate of 50 m
3
of cattle manure plus 750 kg calcium
superphosphate (15.5 % P
2
O
5
) per ha were supplemented,
then the soil of the site was cleared, ploughed, harrowed
and divided into plots. Seeds of squash genotypes were
directly sown in soil. Recommended practices for
disease and insect control were followed.
Leaf area of different genotypes was recorded using a
portable leaf area meter and expressed as (cm
2
). Fruit
weight and fruit length at commercial fresh market
maturity of different squash genotypes were manually
recorded and expressed as (g) and (cm), respectively.
Fruit firmness was measured using a hand Magness
Taylor pressure tester and expressed as (Ib/in2)
(Mitcham et al., 1996). Soluble Solid Content (S.S.C.)
was measured using hand refractometer at 20°and
expressed as percent (%) (Mitcham et al., 1996). The
extraction and determination of ascorbic acid was
performed using the protocol of Pearson (1970) by
titration method using 2,6 dichlorophenolindophenol
in the presence of oxalic acid and expressed as
mg/100 fresh weight (A.O.A.C., 1990).
Statistical analysis:
The experiment was laid-out in a Randomized
Complete Block Design (RCBD) with three replications.
Data were statistically analyzed using Statistica 6
software (Statsoft, Tulsa, Ok, USA) with mean values
compared using Duncanś multiple range with a
significance level of at least p ≤ 0.05.
Genomic DNA isolation, PCR reaction and
RAPD analysis
DNA of 14 squash genotypes of different geographical
origin was extracted from the recent leaves of the
plants as described by Murray and Thompson (1980).
Ten random oligonucleotide (10 mer) primers were
tested for use in RAPD analysis. The primers were
(A01, A02, A03, A04, A05, A06, A07, A08, A09, and
A10) (Laboratories of the Midland Certified Reagent
Company Inc. Texas, USA) (Table 2).
The PCR reaction were carried out in 50 μL volumes
tubes containing 100 ng of genomic DNA, 10 μM of
each primer, 200 μM of dATP, dDTP, dCTP, dGTP, 10
mM Tris-HCL, pH 8.3, 50mMMgCl2 and 0.001% gelatin.
The Taq DNA polymerase (Promega, Corporation,
Madison, WI) concentration was 1.5 units per assay.
The PCR reaction was conducted using Eppendorf
thermocycler programmed according to the following
protocol that consisted of 1 min. at 95 Cºfollowed by
55 cycles of 20 sec. at 94ºC, 30 sec. at 37 ºC, and 2
min. at 72 ºC as described by Nadig et al., (1998).
Amplification products were electrophoresed in 1.5%
Agarose gel in 1 x TAE buffer, stained with ethidium
bromide and visualized with UV transilluminator and
photographed- A 100 bp DNA ladder of 1000 bp
(Promega, Corporation, Madison, WI) was used as a
standard for primers (Gene on, UK).
Analytical procedures
Fragments that were clearly resolved on the gels were
