Fruit Properties and Genetic Diversity of Five Ber (Ziziphus
mauritiana Lamk) Cultivars.
R. S. Obeed, M. M. Harhash and A. L. Abdel-Mawgood
Plant Production Department, College of Food Sciences and Agriculture, King Saud
University, P.O. Box 2460, Riyadh, 11451, Saudi Arabia
The present study was conducted on five ber (Ziziphus mauritiana Lamk)
cultivars (Komethry, Pakstany, Um-sulaem, Toffahy and Peyuan) grown in Saudi
Arabia during 2005 and 2006 seasons. The aim of the present study was to investigate
fruit properties (fruit weight, length, diameter, shape, specific gravity, seed weight,
pulp percentage, total soluble solids (TSS), acidity percentage, TSS/acid, vitamin C
content, and total, reducing, and non-reducing sugars). Peyuan cv. had the heaviest
fruit weight, fruit volume and reducing sugar content however, it was the lowest in
pulp percentage and non-reducing sugars among the five cultivars in both seasons.
Toffahy cv. had highest fruit diameter and seed weight while, had lowest TSS %,
vitamin C and total sugars values. Um-sulaem cv. had highest acidity percentage and
vitamin C content and lowest fruit weight, length and TSS/acid. On the other hand,
Pakstany cv. had highest percentage of both pulp percentage, TSS, total and non-
reducing sugars. Finally, komethry had the longest fruit. The molecular
characterization and fingerprint identification of the ber cultivars was conducted using
the ISSR (Inter-Simple Sequence Repeats) technique. The ISSR technique was able to
uniquely characterize and differentiate between the five ber genotypes. Moreover, the
genetic similarity tree showed that the cultivar Um-slaem is genetically distant from
the other four cultivars, and the two cultivars Pakstany and Komethry were
Key words: fruit properties, ber, Ziziphus mauritiana Lamk, fingerprinting, genetic
Ber (Ziziphus mauritiana Lamk) belongs to the Rhamnaceae family. Of the well–
known species of the genus Ziziphus, ber (Z. mauritiana) is the most common in the
tropical and sub–tropical regions, while Z. jujuba is well known in temperate parts of
the world. These species are indigenous to North Africa, Afghanistan, North India,
Southern China, Malaysia, and Queensland in Australia. However, ber is now widely
distributed and has become naturalized in tropical Africa, Iran, Syria, Sri Lanka and
part of the Mediterranean (Kaarira, 1998). Ber can provide food security, due to
sustained production of the fruit, irrespective of drought, as the tree is drought and
saline tolerant and can grow on poor degraded land (Pareek, 2001). Ber fruits are very
nutritious and usually eaten fresh. Fruits are also eaten in other forms, such as dried,
candied, pickled, as juice, or as ber butter (Maydell, 1986). The fruits are a drupe,
varying from round to elongate and from cherry–size to plum–size depending on
cultivar (Reich, 1991). The ber cultivars were varying in fruit physio–chemical
characteristics. Fruit weight ranged from 3.8 to 39.5 g; fruit length ranged from 1.82
to 5.80 cm, diameter 1.1 to 4.7 cm. Fresh mature ber fruits contains 81 to 97 % pulp
(Chovatia et al., 1993, Jawanda et al., 1981 and Ghosh and Mathew, 2002). Ber pulp
contains 12–23 % TSS, 0.13 – 1.42 % acidity, 3.1 – 14.5 % total sugars, 1.4 – 9.7 %
reducing sugars, 5.6% sucrose, 1.5% glucose, 2.1% fructose and 1.0% starch (Bal,
1992 and Ghosh and Mathew, 2002). Ber pulp is a rich source of vitamin C. Jawanda
and Bal (1978) reported that ascorbic acid content in different ber cultivars ranged
from 39 – 166 mg/100g of pulp.
In recent years, a series of molecular markers techniques have been developed to
analyze and estimate genetic diversity in plant species. Among the various marker
systems, the randomly amplified polymorphic DNA (RAPD) is one of the most
popular DNA-based approaches (Martin and Hernandez Bermejo, 2000; Bekessy et
al., 2002, Abdel-Mawgood et al., 2005 and 2006). The RAPD technique is a
potentially simple, rapid, reliable and effective. In addition, it has the advantage of
that there is no prior knowledge of DNA sequence information is required. However,
there is a major disadvantage associated with it which is the lack of reproducibility
between laboratories (Pooler and Scorza 1995, Weeden et al., 1992). On the other
hand, the ISSR is more reproducible than the RAPD technique and is preferred. The
ISSR markers are generated from single-primer PCR reactions where the primer is
designed from di- or trinucleotide repeat motifs with a 5' or 3' anchoring sequence of
one to three nucleotides (Wolfe and Liston, 1998), without the requirement for prior
sequence information (McGregor et al., 2000). Moreover, ISSR technique producing a
high degree of polymorphism, generating reliable information for DNA analysis with
the necessary sensibility to distinguish among individuals genetically related. ISSR
analysis is technically simpler than many other marker systems. The method provides
highly reproducible results and generates abundant polymorphisms in many systems.
The ISSR technique was successfully applied to study genetic diversity in
Astragalus oniciformi populations (Alexander et al., 2004), Penstemon sp (Wolfe et
al., 1995) and in taxonomic studies of Vigna (Ajibade et al., 2000). In ornamental
species ISSRs have been used in Escandón et al., 2005); Nierembergia (Escandón et
al., 2005), Pandorea sp (Jain et al., 1999) and Chrysantemum (Wolff et al., 1995).
The ISSR strategy was applied to generate fingerprints in newly developed inbred
lines of wheat (Abdel-Mawgood, 2007).
The objectives of this work were to study the fruit properties and genetic diversity
of five ber (Ziziphus mauritiana Lamk) cultivars (Komethry, Pakstany, Um-sulaem,
Toffahy and Peyuan) grown in Saudi Arabia.
MATERIALS AND METHODS
The present study was conducted during 2005 and 2006 seasons in order to study
the fruit properties and genetic diversity of five ber (Ziziphus mauritiana Lamk)
cultivars grown in Saudi Arabia namely; Komethry, Pakstany, Um-sulaem, Toffahy
and Peyuan (local names). Eight-year old orchard trees were budded on (Ziziphus
spina-christi Lamk) rootstock, grown in the Agricultural Research and Experiment
Station (Dirab), College of Food Sciences and Agriculture, King Saud University,
Riyadh, Saudi Arabia.
Fruits were harvested at the first week of April. Random samples of 50 fruits
were harvested from each tested tree (five replicates in each cultivar) for fruit quality
determinations. In each fruit sample, fruit weight; volume, length, diameter, fruit
shape and seed weight were determined. The percentage of total soluble solids (TSS)
was determined in fruit juice using BRX-242 digital refractometer. Juice acidity
percentage (estimated as citric acid equivalent) was determined by titration with
NaOH and phenolphthalein indicator. Vitamin C (Ascorbic acid) was determined by
titration with 2, 6 dichlorophenol-endophenol blue dye and expressed as ascorbic acid
(mg per 100 g pulp). Reducing, non–reducing and total sugars were determined
according to (A.O.A.C., 1986).
DNA was extracted from lyophilized young leave powder. Twenty milligrams of
lyophilized powder was used for genomic DNA extraction using the DNeasy Plant
Mini Kit (Qiagen Inc., Mississauga, ON, Canada) according to the manufacturer's
directions. Extracted DNA was quantified by spectrophotometer followed by dilution
to 25 mg µL–L for ISSR analysis.
The Polymerase Chain Reaction (PCR) for the ISSR (Inter-Simple Sequence
Repeats) was performed in a final volume of 25uL containing 1xTaq polymerase
buffer, 0.5 units of FastStart Taq polymerase (Qiagen), 200 mM of each dNTPs
(Promega, USA), 15 ng of random primer (UBC, 2005), 2.5 mM MgCl2, and 25 mg
of genomic DNA. The reaction mixture was performed using the following cycling
parameters: 1 cycle of 15 min at 94oC, followed by 45 cycles of 30 s at 94oC, 1 min at
50oC, 2 min at 72oC, and a final step of 10 min at 72oC. Amplified PCR products
were separated on 1.5% agarose gel in 1xTBE buffer (100mM Tris-HCl, pH8.0, 83
mM boric acid, 1mM EDTA) at 50 volts. The gels were stained with 0.5 ug/ml
ethedium bromide solution and visualized by illumination under UV light. The sizes
of the amplified products were determined by comparison with 100 bp ladder
(Promega, Wisconsin, USA), Sambrook et al., (1989).
The experimental design was completely randomized blocks. Each cultivar (five
cultivars) involved five replications of 50 fruits each. Statistical analysis was
performed with SAS software package version 6.03 (SAS Institute, 1988). To
examine the genetic relationship between the five cultivars, a dendrogram was
constructed using a UPGMA analysis as implemented by NTSYS-pc, Version 2.02c
(Rohlf, 1997). The PCR data generated from the eight different primers were scored
into 0 and 1. For each genotype, the presence of a band (1) or its absence (0) was
RESULTS AND DISCUSSION
Fruits morphology of the five ber cultivars are clear difference in the
morphological characters (Fig. 1). The fruit quality for five ber cultivars presented in
Tables 1 and 2 during 2005 and 2006 seasons.
Peyuan cultivar had the largest fruit weight (32.69 & 33.3 gm) followed by the
other cultivars: Toffahy (31.72 & 31.32 gm), Komethry (21.51 & 22.61 gm), Pakstany
(14.98 & 15.68 gm) and Um- sulaem (14.26 & 14.72 gm) in both seasons,
respectively. Regarding the average fruit length, Komethry cultivar fruit was the
tallest (5.83& 5.87 cm), while Um-sulaem cv. exhibited shortest (3.31&3.19 cm) and
peyuan was intermediate. However, insignificant difference was found between
Pakstany and Toffahy cultivars. Toffahy cultivar had the highest fruit diameter (4.0
&4.01), while Pakstany had the least diameter (2.48 and 2.44 cm). It was noticed that
Toffahy and Peyuan have a significant different in their diameters compared with
other cultivars. However, cultivars Komethry, Pakstany and Um-sulaem showed no
significant difference in their diameters (2.72, 2.48 and 2.90 cm.) respectively. The
data in Tables 1 and 2 indicated that Komethry had the greatest value of fruit shape
(2.14 and 2.04). This value differs significantly with other cultivars fruit shape. On
the other hand, Toffahy cv. gave the least value of fruit shape (1.01 and 0.99).
Concerning the fruit volume there is a clear difference between the five
cultivars. Toffahy and Peyuan have significantly larger fruit volume than the other
three cultivars (35.53 and 36.33 cm3 respectively). While the cultivar Um-suleam has
the smallest fruit volume (16.85 and 17.22 cm3) in both seasons, respectively. The
highest specific gravity was achieved in fruits of Pakstany cv.(1.05 and 1.08) flowed
by Komethry cv. (1.0 and 1.02), while the lowest specific gravity was found in Um-
suleam cv. (0.84 and 0.86) in both seasons, respectively. For seed weight character,
the cultivar Toffahy and Peyuan showed no significantly difference in seed weight.
However, they differ significantly with the other three cultivars. The heaviest seed
were collected form Toffahi cv. (2.0 gm), while the lightest seeds were from Pakstany
cv. (0.72 gm). In all studied cultivars pulp represent a high percentage of fruits which
exceeded 90%. The cultivars of Komethry, Pakstany and Um-suleam gave greater
pulp percentage than other cultivars. Seemingly, pulp percentage showed a
relationship with fruit weight and seed weight.
Regarding TSS percentage in fruits, it was observed that Pakstany cv. had the
highest percentage (12.55 and 12.83%) while the least TSS percentage was obtained
in Toffahy cv fruits (9.0 and 8.93%) for tow season, respectively. There was no
significant difference in TSS percentage in Kemethry, Um-suleam and Peyuan
cultivars in the both seasons. The fruit of Am-suleam cv. had a significantly high
content of juice acidity percentage compared to the other two cultivars. On the other
hand, Pakstany cv. contained the least percentage of acidity. Data in Table 1 and 2
indicated that fruits of Um-suleam cv. had a significantly higher content of V. C. (135
and 133.5 mg/100g pulp) than other cultivars. There was a significant difference
between the fruits of cultivars in their total sugar. The cultivar Pakstany has a
significantly higher value of percentage of total sugar than the other cultivars, but
lower percentage was obtained in the fruits of the cultivar Toffahy (7.67 and 7.88%).
However, in terms of reduced sugar content the fruits of Peyuan were significantly
higher than other cultivars, while the least percentage of reduced sugar was showed in
the fruits of the cultivar Um-suleam (4.47 and 4.77%). Meanwhile, the greatest
percent of non-reducing sugar was found in fruits of Pakstany cv. and the lowest was
Figure (1): Fruit morphology of the five ber cultivars; A= Komethry, B=
Pakstany, C= Um- sulaem, D= Toffahy and E=Peyuan
Table (1): Fruit properties of five ber cultivars during 2005 growing seasons.
Komethry Pakstany Toffahy Peyuan
Fruit weight 21.51 14.98 31.72 32.69 0.86
Table (2): Fruit properties of five ber cultivars during 2006 growing seasons.
5.83 4.40 3.31 4.04 4.39 0.10
2.72 2.48 2.90 4.00 3.52 0.06
2.14 1.77 1.14 1.01 1.25 0.05
21.50 14.25 16.95 35.50 36.02 0.73
1.00 1.05 0.84 0.89 0.91 0.04
1.12 0.72 0.73 2.09 1.96 0.13
94.81 95.22 94.91 93.40 91.32 0.53
10.75 12.55 10.50 9.00 10.86 0.85
0.48 0.35 0.82 0.62 0.44 0.04
22.52 36.15 12.90 14.58 24.62 1.95
120.63 62.88 135.28 43.20 56.45 2.60
5.37 4.82 4.47 4.83 6.37 0.41
4.04 5.68 3.77 2.85 2.22 0.41
9.41 10.50 8.24 7.67 8.59 0.72
Komethry Pakstany Toffahy Peyuan
22.61 15.68 14.72 31.32 33.03 1.33
5.87 4.34 3.19 3.96 4.41 0.09
2.87 2.44 2.92 4.01 3.53 0.08
2.04 1.79 1.09 0.99 1.25 0.04
The data presented in this study which described the physical and chemical
22.10 14.51 17.22 35.63 36.33 0.87
1.02 1.08 0.86 0.90 0.91 0.03
1.17 0.72 0.73 1.98 1.96 0.13
95.00 94.00 95.00 93.75 91.00 1.15
10.60 12.83 9.83 8.93 10.86 0.87
0.50 0.36 0.83 0.61 0.43 0.05
21.15 35.94 11.93 14.67 25.48 3.26
120.15 65.00 133.50 43.95 57.90 2.08
5.77 5.12 4.77 5.03 6.55 0.52
4.34 5.75 3.97 2.85 2.48 0.47
10.01 10.87 8.64 7.88 9.03 0.76
properties of fruits of different ber cultivars were in the range obtained by previous
studies. For example the findings of fruit weight are in line with those reported by
Chovatia et al., (1993) and Jawanda et al., (1981). The measurement of fruit length in
the current study is in agreement with those reported by Morton (1987), Pareek, and
Sharma (1991). However, fruit pulp and chemical contents measurement were varied
than those reported earlier. For instant, in the current study, ascorbic acid content was
ranged from 43 to 135. These results agree with those found by Jawands and Bal
(1978). The total sugar content and reducing sugar were in agreement with the data
mentioned by Bal (1992) and Ghosh and Mathew (2002).
There is a complete lack of information on the extent of genetic diversity in ber
(Singh et al., 2006). For our knowledge, this is the first study to investigate the extent
of genetic diversity of cultivated ber genotypes in Saudi Arabia. Total of 15 ISSR
primers have been used in this study. Out of which only 8 ISSR primers gave good
polymorphism with reproducible pattern. The sequence of the primers, the number of
bands scored for each primer, the number of polymorphic bands and the percentage of
polymorphism is described in Table 3. All the eight primers produced polymorphic
bands. Two primers gave 100% polymorphism. The two primers are P2 (AGAG)4T
and P7 (CTCT)4T; they also gave a pattern that can be used for fingerprinting to
distinguish between the five cultivars. The rest of the primers gave polymorphism
ranged from 25% to 70%. The gel patterns using the ISSR primer (P2) (AGAG)4T are
given in Fig 3.
The percentage of polymorphism as an average of the eight primers is 53%. However,
in another study (Singh et al., 2006) using 11 primer pairs and AFLP technique, the
percentage of polymorphic bands was 84%. The difference between the two studies in
the percentage of polymorphic alleles could be due to the using of different genotypes
and that our genotypes are cultivated which always have a less genetic diversity than
the wild types (Singh et al., 2006), this is an addition to the difference in both the
ISSR and AFLP in showing the polymorphism.
The genetic similarity of tree produced from the above data is presented in figure 2.
The cultivar Um-Sulaem was genetically distant from the other four cultivars with
genetic similarity of 60%. The other four cultivars have genetic similarity coefficient
close to 65.5%. The second branch was represented by the cultivar Toffahy and the
rest of the three cultivars. The third branch was represented by Komethry, Pakstany
and Payuan cvs. The two cultivars Komethry and Pakstany were very closely related
with genetic similarity of 76%. The only published results so far on ber genetic
diversity were by (Singh et al., 2006), who used the AFLP technique to study genetic
diversity in 33 cultivated ber accessions. The genetic diversity index between the 33
accessions ranged from 0.14 to 0.86 with an average of 0.62. They also stated that the
genetic diversity between the cultivated species is a lot less than that in the wild
relatives that are used as a rootstock. Their finding is in agreement with our data,
which showed that genetic similarity between the cultivars under this study is high.
As indicated above the cultivar Um-Sulaem was genetically distant from the rest of
the cultivars. It has the lowest value of fruit weight and length, seed weight, TSS/acid
and reducing sugars. However, it has the highest vitamin C content, pulp percentage
and acidity percentage. On the other hand, although both the Komethry and Pakstany
cultivars were very closely related to each other based on the genetic tree, although
there was no similarity between the two cultivars in their fruit properties.
Table (3). ISSR primer used in this study, number of bands from each primer,
polymorphic bands and percentage of polymorphism.
No Primer Sequence (5'- 3') No. of Bands
1 ATA TAT ATA TAT ATA TT 14 7 50
2 AGA GAG AGA GAG AGA GT 10 10 100
3 AGA GAG AGA GAG AGA GC 10 7 70
4 AGA GAG AGA GAG AGA GG 8 5 62.5
5 GAG AGA GAG AGA GAG AT 10 3 30
6 GAG AGA GAG AGA GAG AA 12 3 25
7 CTC TCT CTC TCT CTC TT 10 10 100
8 CTC TCT CTC TCT CTC TA 8 7 87.5
Figure 2: Genetic similarity coefficient between the five ber cultivars
Figure 3: Polymorphism based on the ISSR of the five genotypes using the ISSR
primer (AGAG)4T. M, is 100bp molecular weight marker. lane 1 is Komethry, 2,
Toffahy, 3, is Payuan, 4 is Um-Sulaem and 5 is Pakstany.
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