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JKAU: Met., Env. & Arid Land Agric. Sci., Vol. 20 No. 1, pp: 63-79 (2008 A.D./1429 A.H.)
63
Variation in Productive Characteristics and Diversity
Assessment of Garlic Cultivars and Lines Using DNA
Markers
S. Al-Otayk, M. Z. El-Shinawy 1 and M. I. Motawei
Department of Plant Production and Protection, College of Agriculture
and Veterinary Medicine, Al-Qassim University, Saudi Arabia, and
1 Horticulture Dept., Faculty of Agric., Ain Shams Univ., Cairo, Egypt
Abstract. Garlic cultivars Egyptian, Chinese, and Elephant, and sex
Chinese lines were evaluated for their productivity in two field
experiments during 2004/2005 and 2005/2006 winter seasons. The
results indicated that the maximum plant height was 70.5 and 70.8 cm
for Egyptian cultivar in both seasons, respectively. The maximum
value of leaves area per plant were observed with Elephant cultivar and
Chinese line (L2). Elephant cultivar was superior on the other cultivars
and lines in plant fresh weight, while, the Chinese line (L4) had the
maximum plant dry weight and the highest chlorophyll content. Bulb
fresh and dry weight of Egyptian cultivar (Balady) was the lowest
among the cultivars and lines tested. On the other hand, Egyptian
cultivar (Balady) produced more cloves number compared with the
other cultivars and Chinese lines. Elephant and Chinese cultivars gave
the highest mean values of bulb diameter compared with Egyptian
cultivar. Also, Chinese lines (L2 and L5) gave higher mean values of
bulb diameter compared with the other Chinese lines. The same trend
was observed for clove weight. Total soluble solid (TSS) content of
cloves was highest in Egyptian cultivar (Balady) and Chinese line 5.
Marketable yield (g/m2) showed that Elephant cultivar and Chinese line
5 (L5) produced the highest yield, while Egyptian cultivar (Balady) and
Chinese line 6 (L6) produced the lowest yield.
Two types of molecular markers, random amplified polymorphic
DNA (RAPD) and inter-simple sequence repeat (ISSR), were assayed
to determine the genetic diversity of sex garlic lines and three garlic
cultivars. A high level of polymorphism among garlic cultivars and
lines was found with both RAPD and ISSR markers, while, ISSR
revealed higher polymorphism among Chinese lines than RAPD. The
UPGMA dendrogram generated from RAPD data clearly indicated four
S. Al-Otayk, et al.
64
main clusters. The dendrogram generated from ISSR data clearly
indicated five clusters. Chinese line (L6) was separated from the other
Chinese lines and this line gave the lowest yield and total soluble solid
content compared to the other lines and Chinese cultivar. Polymorphic
ISSRs are abundant in garlic and demonstrated genetic diversity among
related lines. Therefore, ISSR is an additional tool for fingerprinting
and detailed assessment of genetic relationships in garlic.
Keywords. Garlic cultivars and lines, productive characters, fingerprint,
genetic diversity, RAPD markers, ISSR markers.
Introduction
Garlic (Allium sativivum L.) is a perennial plant whose bulb is
economically important as a food additive. Most garlic plants are sterile
and vegetatively propagated by cloves. The sterility of garlic could be due
to the structural heterozygosity of chromosomes, though its definite cause
is uncertain (Etoh, 1985). Garlic has a large and complex genome with two
pairs of satellite chromosomes in the basic karyotype (Kim and Seo, 1991;
Lee, et al., 2003). It is an unusual crop in that, despite being exclusively
propagated asexually over centuries, it maintains a diverse phenotype
amongst different clones. This makes garlic an ideal species for
investigating heritage and diversity. Using molecular techniques scientists
are able to evaluate diversity between strains. Through the development of
a phylogenetic tree, diversity is established between clones based on their
relative position within the tree. Mutations such as single base
substitutions, inversions, or deletions allow for phylogenetic
differentiation. A tree is then useful for breeders to select diverse parents
in making crosses for the development of superior crops. Also, germplasm
from a country of origin may help to aid in the selection of appropriate
growing climates. The premise behind this is that a selection will have
developed in adaptation to a particular climate, making it better suited to
similar climates (Fernandez, et al., 2003). Prior studies have used total
genomic DNA to screen for molecular markers by employing such method
as RAPD’s (random amplified polymorphic DNA) (Maas and Klaas,
1995; Nabulsi, et al., 2001 and Ipeck, et al., 2003). Moreover, several
PCR-based DNA fingerprinting techniques, including simple sequence
repeat (SSR), and amplified fragment length polymorphism (AFLP) are
available for detecting genetic differences within and among cultivars
(Volk, et al., 2004). Among these, simple sequence repeat (SSR) markers
are efficient, cost-effective and can detect a significantly higher degree of
polymorphism in onion (Kuhl, et al., 2003). They are ideal for genetic
Variation in Productive Characteristics… 65
diversity studies and intensive genetic mapping. An alternative method to
SSR, called inter-SSR (ISSR)-PCR (Nagaoka and Ogihara, 1997), has also
been used to fingerprint the different plant species and cultivars (Nagaoka
and Ogihara, 1997; Levi and Rowland, 1997; Wolf, et al., 1998; Nagaraju,
et al., 2002 and Al-Humaid, et al., 2004).
The objectives of this study were to: (1) evaluate garlic cultivars and
lines selected from Chinese cultivar for the productivity and (2)
investigate the assessment of genetic diversity in garlic cultivars and lines
using RAPD and ISSR markers.
Materials and Methods
Plant Materials and Field Experiments
Field experiments were conducted at the Experimental Farm of the
College of Agriculture and Veterinary Medicine, Al-Qassim University.
Three garlic cultivars, namely Egyptian (Balady), Elephant, and Chinese,
and six lines of Chinese garlic were planted in 13th and 8th October in 2004
and 2005 in the first and second seasons, respectively. Cloves of garlic
cultivars and lines were planted at 7 cm spacing of both sides of ridges
spaced 60 cm apart. The plants were fertilized at the rate of 300 kg of
ammonium sulphate (20.5% N), 300 kg of calcium superphosphate
(15.5% P2O5) and 200 kg potassium sulphate (48% K2O) per fedddan.
Drip irrigation was used.
The soil type of this farm is classified as a sandy soil. Data in Table 1
shows specific soil characteristics. The layout of the experiments was
completely randomized design with four replicates.
Table 1. The soil mechanical analysis of experimental site.
Mechanical analysis
Bulk density
g /cm3
Water
holding
capacity (%)
Field
capacity (%)
Wilting point
(%)
Sand Silt Clay
96.3 % 1.8 % 1.9 % 1.501 17.17 9.6 4.35
S. Al-Otayk, et al.
66
Measurements
Four plants were randomly chosen for recording the vegetative growth
parameters which included, plant height (cm), number of leaves per plant,
leaves area /plant (cm2), plant fresh and dry weight. Also, chlorophyll
content was measured of the fifth upper leaf using Minolta chlorophyll
Meter SPAD –501.
At the harvesting stage four plants were randomly chosen for
recording yield parameters which included, bulb fresh and dry weight (g),
clove fresh and dry weight (g), bulb diameter (cm), number of cloves, and
total soluble solid (T.S.S) (%). Also, marketable yield per square meter
were recorded.
DNA Extraction
Bulk leaf samples from garlic lines and cultivars were used. The bulk
sample of leaves was first ground into fine powder with liquid nitrogen.
DNA was extracted in 10 ml of CTAB buffer consisting of: 50 mM NaCl,
10 mM Tris-HCl pH 7.5, 5 mM EDTA, and 1% CTAB. The homogenate
was incubated for 2 hours at 65 ºC with occasional mixing. Following
incubation, 5 ml of chloroform/isoamylalcohol (24:1) were added to the
tubes, mixed, and centrifuged at 260 g for 10 min. The aqueous phase was
removed to a fresh tube and an equal volume of ice-cold isopropanol was
added followed by centrifugation as above to precipitate the DNA. The
pellet was dissolved in TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM
EDTA). The DNA concentration was assessed spectrophotometrically at
260 nm, and quality was assessed by the 260/280 ratio (Sambrook, et al.,
1989). The DNA was suspended to a final concentration of 5 ng/l in 0.5X
TE and stored at 4°C.
RAPD Analysis
A total of twenty 10-mer oligonucleotides with arbitrary sequence
from Operon (Table 2) were used in RAPD analysis. The PCR reaction
mixture consisted of 50 ng genomic DNA, 1×PCR buffer, 2.0 mmol/l
MgCl2, 100 µmol/l of each dNTP, 0.1 µmol/l primer and 1U Taq
polymerase in a 25µl volume. The amplification protocol was 94 ºC for 4
min to pre-denature, followed by 45 cycles of 94 ºC for 1 min, 36 ºC and
72 ºC for 1 min, with a final extension at 72 ºC for 10 min. Amplification
products were fractionated on 1.5% agarose gel.
Variation in Productive Characteristics… 67
Table 2. Plant height, number of leaves and leaf area of garlic cultivars and lines during
2004/05 and 2005/06 seasons.
Plant height (cm) No. of leaves/plant Leaf area /plant (cm2)
Cultivars 2004/05 2005/06 2004/05 2005/06 2004/05 2005/06
Line 1 (L1) 61.0 c 62.5 b 8.8 ab 7.8 ab 256.6 bc 262.9 f
Chinese garlic (Ch) 54.5 d 57.2 d 8.5 ab 8.0 ab 226.8 bc 235.3 g
Line 2 (L2) 60.5 c 60.5 c 9.3 a 8.5 a 353.2 b 378.1 b
Line 3 (L3) 60.8 c 58.0 d 7.8 bc 7.0 b 198.3 c 212.2 h
Line 4 (L4) 64.3 b 63.0 b 8.3 abc 8.0 ab 350.8 b 350.6 c
Line 5 (L5) 59.3 c 60.0 c 9.3 a 8.5 a 346.4 b 340.9 d
Line 6 (L6) 69.3 a 63.3 b 8.3 abc 7.5 ab 281.9 bc 284.5 e
Egyptian garlic (B) 70.5 a 70.8 a 7.3 c 7.5 ab 244.9 bc 146.6 i
Elephant garlic (El) 53.8 d 51.5 e 8.8 ab 8.5 a 517.9 a 535.4 a
-Data are expressed as mean
-Means within the same column and followed by the same coefficient are not significant different from each
other (p ≤ 0.05).
ISSR Assay
The ISSR-PCR method was carried out according to Negaoka and
Ogihara, (1997). Amplification were carried out in 25 μl reaction volumes,
containing 1X Taq polymerase buffer (50 mM KCl, 10mM Tris, pH 7.5,
1.5 mM MgCl2) and 1 unit of Taq polymerase (Pharmacia Biotech,
Germany) supplemented with 0.01% gelatin, 0.2 mM of each dNTPs
(Pharmacia Biotech, Germany), 50 ρmol of ISSR primers (Table 3), and
50 ng of total genomic DNA. Amplification was performed in a thermal
cycler (Thermolyne Amplitron) programmed for 1 cycle of 2 min at 94˚C;
and 35 cycles of 30 secs at 94˚C, 45 secs at 44˚C, and 1.3 min at 72˚C;
followed by 20 min at 72˚C.
After completion of PCR, samples were cooled immediately to 10ºC
and stored at 4ºC until gel separation. A gel-loading solution (5 µl) was
added, and 10 µl of the total product volume was resolved in 1.5% agarose
in 1X TAE buffer for 2 h aside with a 100- bp ladder (Pharmacia,
Germany) as the size standard. Gels were stained in ethedium bromide and
images were recorded.
S. Al-Otayk, et al.
68
Table 3. Plant fresh and dry weight and chlorophyll content of garlic cultivars and lines
during 2004/05 and 2005/06 seasons.
Plant fresh weight ( g)
Plant dry weight ( g) Chlorophyll
(Value )
Cultivars &
Lines
2004/05 2005/06
2004/05 2005/06 2004/05 2005/06
Line 1 (L1) 134.5f 130.4 f 43.5 b 40.0 e 61.7 f 62.5 f
Chinese garlic 142.3 e 143.8 e 44.3 b 45.8 c 66.1 d 66.0 d
Line 2 (L2) 167.9 c 165.4 c 55.0 a 52.5 b 64.5 e 64.5 e
Line 3 (L3) 160.5 d 160.3 d 52.1 a 51.1 b 69.8 c 71.0 c
Line 4 (L4) 171.9 c 170.3 b 56.0 a 55.5 a 71.9 b 75.6 b
Line 5 (L5) 147.2 e 145.4 e 45.1 b 44.2 cd 70.8 bc 70.6 c
Line 6 (L6) 164.9 cd 163.0 cd 52.9 a 53.5 ab 69.5 c 72.1 c
Egyptian garlic 178.2 b 171.2 b 53.5 a 53.8 ab 75.0 a 76.6 a
Elephant garlic 186.8 a 183.9 a 40.1 c 41.9 de 69.9 c 70.6 c
-Data are expressed as mean
-Means within the same column and followed by the same coefficient are not significant different from each
other (p ≤ 0.05).
Data Analysis
Data were statistically analyzed by using a randomized complete
block design with three replicates according to Snedecor and Cochran
(1980). The two growing seasons were analyzed separately. The least
significant differences (LSD) test was used to compare means at the 5%
level. Only differences significant at P≤0.05 are considered in the text.
Data of RAPD and ISSR analysis were scored for computer analysis
on the basis of the presence or absence of the amplified products for each
ISSR primer. If a product was present in a cultivar, it was designated “1”,
if absent it was designated “0”. Pair-wise comparisons of cultivars, based
on the presence or absence of unique and shared polymorphic products,
were used to generate similarity coefficients based on SIMQUAL module.
The similarity coefficients were then used to construct a dendogram by
UPGMA (Unweighted Pair-Group Method with Arithmetical Averages)
using NTSYS-PC software version 2.0 (Exeter Software, New York)
(Rohlf, 2000).
Results and Discussion
Growth and Yield Characters
There were differences in plant height, number of leaves and leaves
area per plant among garlic cultivars and lines (Table 2). The maximum
plant height was 70.5 and 70.8 cm for Egyptian cultivar in both seasons,
Variation in Productive Characteristics… 69
respectively. Hussein, et al. (1995) found that the maximum plant height
for Egyptian cultivar was 74.0 cm. Whereas, Omer and Abou Hadid
(1992) reported approved approximately 105.5 cm for Egyptian cultivar.
Concerning the leaves number, it can be noted that Chinese line (L5) (9.3
and 8.5) had more leaves number compared with Egyptian cultivar
(Balady) (7.3 and 7.5) in both seasons, respectively. The maximum value
of leaves area per plant were observed with Elephant cultivar and Chinese
line (L2). Elephant cultivar was superior than the other cultivars and lines
in plant fresh weight (186.8 and 183.9 g) in both seasons, respectively.
While, the Chinese line (L4) had the maximum plant dry weight (56.0 and
55.5 g) in both seasons, respectively (Table 3). Also, the last line and
Egyptian cultivar had the highest chlorophyll content of the tested
cultivars and lines in both seasons (Table 3).
For bulb fresh weight, it is apparent in Table 4 that Elephant cultivar
and Chinese line (L5) produced the highest bulb fresh weight (117.6 and
118.6 g) and (68.7 and 67.6 g) in both seasons, respectively. Also, Chinese
lines (L2 and L5) produced the highest bulb dry weight. Bulb fresh and dry
weight of Egyptian cultivar (Balady) was the lowest among the cultivars
and lines tested. On the other hand, Egyptian cultivar (Balady) produced
more cloves number (32.0 and 29.5) compared with the other cultivars and
Chinese lines in both seasons, respectively. These results are in agreement
with Omer and Abou Hadid (1992) who reported that Chinese cultivars
produced larger bulbs in comparison with Egyptian cultivars.
Table 4. Bulb fresh and dry weight and number of cloves of garlic cultivars and lines during
2004/05 and 2005/06 seasons.
Bulb fresh weight ( g) Bulb dry weight ( g) No. of cloves
Cultivars &
Lines 2004/05 2005/06 2004/05 2005/06 2004/05 2005/06
Line 1 (L1) 49.9 f 52.7 ef 19.1 e 20.0 d 13.5 b 14.0 b
Chinese garlic 54.7 e 55.2 e 24.9 c 22.4 c 8.5 ef 9.0 ef
Line 2 (L2) 62.7 c 63.3 c 34.9 a 32.6 a 7.8 f 8.0 f
Line 3 (L3) 51.6 f 50.9 f 20.7 d 20.8 d 10.0 d 9.5 d
Line 4 (L4) 57.03d 58.4 d 21.5 d 21.1 d 9.3 de 9.3 de
Line 5 (L5) 68.7 b 67.6 b 26.9 b 24.4 b 11.3 c 11.8 c
Line 6 (L6) 37.2 g 36.9 g 13.7 f 13.1 e 11. 0 c 10.5 c
Egyptian garlic 36.5 g 36.2 g 11.7 g 21.1 e 32.0 a 29.5 a
Elephant garlic 117.6 a 118.6 a 23.8 c 24.6 b 5.3 g 4.8 g
-Data are expressed as mean
-Means within the same column and followed by the same coefficient are not significant different from each
other (p ≤ 0.05).
S. Al-Otayk, et al.
70
The character of bulb diameter is among the major harvestable yield
components that contribute the ultimate development of different cloves
order, number and diameter of arranged cloves attached the disc steam
structure. In this respect the data in Table 5 illustrated that Elephant and
Chinese cultivars gave the highest mean values of bulb diameter compared
with Egyptian cultivar. Also, Chinese lines (L2 and L5) gave higher mean
values of bulb diameter compared with the other Chinese lines. The same
trend was observed for clove weight, where Elephant cultivar and Chinese
line (L2) gave the highest mean values of cloves fresh and dry weight
compared with the other cultivars and lines.
Table 5. Bulb diameter, and cloves fresh and dry weight of garlic cultivars and lines during
2004/05 and 2005/06 seasons.
Bulb diameter (cm) Cloves fresh weight
( g)
Cloves dry weight
( g )
Cultivars &
Lines
2004/05 2005/06 2004/05 2005/06 2004/05 2005/06
Line 1 (L1) 5.4 c 5.7 c 3.3 d 1.4 f 3.3 d 1.4 d
Chinese garlic 6.2 b 5.9 b 6.2 c 2.7 c 5.4 cd 2.9 b
Line 2 (L2) 6.7 b 5.9 b 7.7 b 4.0 b 7.8 b 4.5 a
Line 3 (L3) 5.5 c 5.3 d 4.9 c 2.2 d 5.0 d 2.0 c
Line 4 (L4) 5.1 d 5.9 b 5.9 c 2.2 d 5.9 c 2.3 c
Line 5 (L5) 6.0 b 6.0 b 5.5 c 1.9 e 5.3 cd 2.3 c
Line 6 (L6) 5.1 d 5.1 e 2.9 d 1.2 f 3.4 d 1.2 d
Egyptian garlic 5.1 d 5.1 e 1.0 e 0.4 g 1.1 e 0.4 e
Elephant garlic 7.4 a 7.2 a 19.3 a 5.2 a 24.0 a 4.5 a
-Data are expressed as mean
-Means within the same column and followed by the same coefficient are not significant different from each
other (p ≤ 0.05).
Total soluble solid (TSS) content of cloves was highest in Egyptian
cultivar (Balady) and Chinese line 5 (L5) (37.0 and 38.6 %) and (38.8 and
38.4 %) in both seasons respectively (Table 6). On the other hand,
Elephant cultivar and Chinese line 6 (L6) gave the lowest total soluble
solid content. Singh and Chand (2003) found that total soluble solid
content of cloves was significantly differed among garlic cultivars and
clones. Marketable yield (g/m2) showed that Elephant cultivar and
Chinese line 5 (L5) resulted in the highest yield (2352.1 and 2372.5
g/m2) and (1373.2 and 1352.2 g/m2) in both seasons, respectively
(Table 6). While, Egyptian cultivar (Balady) and Chinese line 6 (L6)
produced the lowest yield (729.8 and 723.2 g/m2) and (743.3 and 714.9
g/m2) in both seasons, respectively. Similar results were reported by
Hussein, et al. (1995) who found that Balady cultivar produced the
lowest garlic yield.
Variation in Productive Characteristics… 71
Table 6. T.S.S and marketable yield of garlic cultivars and lines during 2004/05 and 2005/06
seasons.
Cloves T.S.S % Marketable yield g /m2
Cultivars & Lines
2004/05 2005/06 2004/05 2005/06
Line 1 (L1) 37.3 b 38.3 a 999.1 f 1055.2 b
Chinese garlic 35.6 bc 34.9 c 1095.9 e 1104.8 e
Line 2 (L2) 36.1 bc 36.8 ab 1254.7 c 1265.2 c
Line 3 (L3) 34.5 cd 35.1 c 1032.4 f 1018.2 f
Line 4 (L4) 36.0 bc 35.5 bc 1140.6 d 1167.6 e
Line 5 (L5) 38.8 a 38.4 a 1373.2 b 1352.2 b
Line 6 (L6) 33.2 d 32.7 d 743.3 g 714.9 g
Egyptian garlic 37.0 b 38.6 a 729.8 g 723 1 g
Elephant garlic 26.8 e 27.8 c 2352. 0 a 2373.4 a
-Data are expressed as mean
-Means within the same column and followed by the same coefficient are not significant different from
each other (p ≤ 0.05).
Genetic Diversity
For RAPD analysis, random primers (Operon Technologies Alameda,
Calif.) reported to be polymorphic in previous studies for garlic (Maas and
Klaas, 1995; Ipek, et al., 2003) were tested. Eight primers of arbitrary
nucleotide sequence were used to amplify DNA segments from garlic
cultivars and lines. The number of amplification bands per primer varied
between 5 and 11. Analysis of the 8 primers among garlic cultivars and
lines included in this study generated 62 bands, 52 of which were
polymorphic among garlic cultivars and lines (Table 7).
Table 7. RAPD primers with the number of amplified products, polymorphic fragments
among garlic cultivars and lines, and polymorphic fragments among Chinese lines.
Operon primers Amplified
products
Polymorphic
Fragments among garlic
cultivars and lines
Polymorphic fragments
among Chinese lines
OPA-01
OPA-13
OPF-03
OPF-04
OPF-05
OPF-06
OPF-07
OPF-08
11
6
6
6
12
5
7
9
10
4
4
6
10
4
5
9
1
0
0
0
1
1
0
1
S. Al-Otayk, et al.
72
There were 6.5 polymorphic bands per primers in average, while,
analysis of RAPD primers among Chinese garlic lines generated only 4
polymorphic bands. Ipek, et al. (2003) found that all garlic clones shared
100% of RAPD bands within each group. Examples of polymorphism are
shown in Fig.1.
OPF-3
M L1 Ch L2 L3 L4 L5 L6 El B
OPF-5
Fig. 1. Polymorphism revealed using primer OPF-3 and OPF-5 to amplify genomic DNA
purified from the tested garlic cultivars and lines. M lane is 1 kbp ladder DNA
marker.
The UPGMA dendrogram generated from RAPD data clearly
indicated four main clusters (Fig. 2). The first cluster contained Chinese
lines L1, L2, and L3. The second cluster contained Chinese cultivar and
M L1 Ch L2 L3 L4 L5 L6 El B
Variation in Productive Characteristics… 73
lines L4, L5 and L6. The Chinese line 4 was genetically closed to Chinese
cultivar. The third cluster contained Egyptian cultivar (Balady) (Allium
sativum). The fourth cluster contained Elephant garlic (Allium
ampeloprasum). Etoh, et al. (2003) found that the genetic similarity
among the Iberian garlic clones using RAPD marker was high, and poor
genetic diversity was estimated among the clones from Spain and Portugal,
while the genetic similarity among the Central Asian clones was
comparatively low, and greater genetic diversity was estimated among
those Central Asian clones.
Fig. 2. Dendrogram constructed from similarity coefficients and showing the clustering of the
tested garlic cultivars and lines using RAPD markers.
In ISSR analysis, the number of amplification bands per primer varied
between 0 and 10. Examples of polymorphism are shown in Fig. 3. The
trinucleotide repeats (CTC)n primer had more bands than (CAC)n (CTC)n
and (GTG)n primers, and dinucleotide repeats (CA)n primer (Table 8).
S. Al-Otayk, et al.
74
M L1 Ch L2 L3 L4 L5 L6 El B
D24
M L1 Ch L2 L3 L4 L5 L6 El B
HB14
Fig. 3. Polymorphism revealed using primer D24 and HB14 to amplify genomic DNA
purified from the tested garlic cultivars and lines. M lane is 1 kbp ladder DNA
marker.
Also, among seven ISSR primers, poly (CTC) based primers accounted
37.5% of total polymorphic bands among garlic cultivars and lines.
Analysis of ISSR primers among Chinese garlic lines generated six
polymorphic bands. Therefore, ISSR revealed higher polymorphism
among Chinese lines than RAPD. Mondal, et al. (2008) found that a total
of 17 selected RAPD and 21 ISSR primers produced 119 and 153 bands
respectively, of which 56 and 114 were polymorphic correspondingly. Of
the two markers, ISSR revealed higher polymorphism (74.5%) than RAPD
(47.1%) in peanut genotypes.
Variation in Productive Characteristics… 75
Table 8. ISSR primers with the number of amplified products and polymorphic fragments
among garlic cultivars and lines, and polymorphic fragments among Chinese lines.
Primers Sequence
5` to 3`
Amplified
products
Polymorphic
Fragments among garlic
cultivars and lines
Polymorphic
fragments among
Chinese lines
P02
D12
D14
D24
HB 13
HB 14
HB 15
(ATCG)4
(GA)6CG
(CAC)3GC
(CA)6CG
(GAG)3GC
(CTC)3GC
(GTG)3GC
0
0
4
4
6
10
4
0
0
2
3
3
6
2
0
0
1
1
0
2
2
The dendrogram generated from ISSR data clearly indicated five
clusters (Fig. 4). The first cluster included Chinese lines 1, 2, 4, and 3. The
second cluster included Chinese cultivar and line (L5). The third cluster
contained Chinese line (L6). The fourth cluster contained Egyptian
cultivar (Balady) (Allium sativum). The fifth cluster contained Elephant
garlic (Allium ampeloprasum). It should be noted that Chinese line (L6)
Fig. 4. Dendrogram constructed from similarity coefficients and showing the
clustering of the tested garlic cultivars and lines using ISSR markers.
S. Al-Otayk, et al.
76
was separated from the other Chinese lines and this line gave the lowest
yield and total soluble solid content compared to the other lines and
Chinese cultivar. Bradley, et al. (1996) reported that bolting and
intermediate/nonbolting garlic forms could be separated from each other
based on cluster analysis of RAPD markers. In conclusion, ISSR
technology is a useful tool for analysis of genetic diversity of garlic along
with productive characters and RAPD markers. ISSR markers can provide
a better approximation to true variation among garlic lines.
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