Journal of Nuts 6(2):155-164, 2015
ISSN: 2383 - 319x
Identification and Screening of Homozygous and Heterozygous Almond Progenies from
Self-Pollinated Touno Cultivar Using PCR
P.Najafi1, A. Imani*2, S.M. Miri 1, M. Zinalabdini3
1
Department of Horticulture, Karaj Branch, Islamic Azad University, Karaj, Iran
2
Department of Horticultural, Seed and Plant Improvement Iinstitute (SPII), Karaj, Iran
3
Department of Molecular Genomics, Agricultural Biotechnology Research Institute (ABRII), Karaj, Iran
Received: 23 June 2015
Accepted: 7 October 2015
Abstract
Self-incompatibility is one of the most important difficulties in almond production which reduce fruit set dramatically
and makes orchard management difficult. Therefore, breeding almond to produce self-compatible genotypes is very
important. In this research identification and screening of 86 almond progenies obtained from selfing Touno after the selfpollination by PCR reaction with specific primers of CEBASf and AS1. PCR results confirmed the situation of selfcompatible hybrids. In addition, it indicated that, frequencies of Sf, and S1 was 100% and 50% in progenies respectively.
Self-compatible hybrids had been identified that can be used in almond breeding programs particularly to development the
monoculture of almond orchards. So to identify and screening homozygous self-compatibility almonds be capable of be
another step towards creating monoculture of almond and use in breeding programs further.
Keywords: Almond, Hybrid, Self-compatible, Self-pollination
Introduction
Almond is a temperate zone fruit tree that is
gametophytic type of self-incompatibility, which means
cultivated in many countries because of its nutritional
that there is no pollen germination on the stigma
value. Although the almond is one of the oldest crops
(Yamashita et al., 1987), or that tube growth stops, most
used by humans, its specific environmental requirements
often in the upper third of the style (Socias i Company et
have restricted its commercial production to specific
al., 1976; Mousavi et al., 2014). Self-incompatibility in
areas of the world (Kester and Gradiziel, 1996). Self-
almond is controlled by a single S-locus with multiple
incompatibility is a wide- spread and heritable
codominant alleles (Socias I Company et al., 1976).
reproductive phenomenon in flowering plants, in which
This trait is expressed in the style by special
self-fertilization is prevented by rejection of pollen from
glycoproteins (S-RNases) that arrest the growth of
the same plant. This is an evolutionary advantage
pollen tubes in self-incompatible cultivars (Socias I
because of its effectiveness in avoiding inbreeding and
Company et al., 1976; Boskovic et al., 1998; Boskovic
the encouragement of out-crossing (Socias i Company,
et al., 2003).
1990; Alonso and Socias i Company, 2005a). Prunus
species, such as almond, are characterized by a
*Corresponding author: Email: [email protected]
155
Journal of Nuts 6(2):155-164, 2015
Nowadays, the use of molecular methods is very
in the cultivars Genco, Tuono and Ferragnes (Ortega
important for identification of self-compatible and self-
and Dicenta, 2008). So far, 35 S incompatibility alleles,
incompatible cultivars. In addition, these methods are
in addition to the Sf self-compatibility allele, have been
very reliable for determination of S-genotypes in almond
identified in almond using different molecular analyses,
(Alonso and Socias I Company, 2006; Mousavi et al.,
specifically, ribonucleasis, S allele PCR and sequencing
2011). So far, it has been shown that there are about 35
analysis (Lopez et al., 2006). More than 154 almond
self-incompatible alleles (S1, S2,….S35) and one self-
cultivars have been genotyped (Lopez et al., 2006) and
compatible allele (Sf) (Lopez et al., 2006; Boskovic et
19 cross-incompatible groups have been established.
al., 2007; Mousavi et al., 2011). Only a limited number
The techniques used to identify genotypes as self-
of the numerous almond cultivars grown worldwide are
compatible can be more pointed; use of paper bags and
self-compatible, the majority of which come from the
flower covers in branches that contain flower and finally
Italian region of Apulia (Kester and Gradiziel, 1996).
examine the fruit set (Socias i Company, 1990),
Prominent among these are Tuono and Genco, Filippo
evaluation of pollen tube growth in pistils using
Ceo
Mazzetto
fluorescence microscopy (Mousavi et al., 2014), and use
synonymous for Tuono (Lopez et al., 2006), Falsa
of molecular methods including PCR (Lopez et al.,
Barese, Ferrante and Palatina (Godini, 2002). As these
2004). The PCR method to detect genotypes has been
cultivars were shown to be capable of transmitting their
used by researchers such as Socias i Company (1990),
self-compatibility to their offspring (Socias I Company
Channuntapipat et al. (2001), Martin-Gomez (2003),
and Felipe 1988) using them has proved to be the most
Channuntapipat et al. (2003) and Alonso and Socias i
effective method for obtaining new self-compatible
Company (2006). Based on these results, main objective
cultivars. Knowledge of the inheritance of self-
of this study was identification and screening of
compatibility is an essential step in the attainment of
homozygous (SfSf) from heterozygous (S1Sf) almond
such an objective (Socias I Company et al. 1976). Duval
progeny hybrids obtained from self-pollination of Touno
et al. (2001) analyzed a progeny of 'Ferralise x Tuono'.
using PCR technique.
(Kester
and
Gradiziel,
1996),
'Ferralise' shares the S1 allele with 'Tuono' (S1Sf) and has
the same S1S3 genotype 'Ferragnès'. All tested progeny
seedlings, except one, were genotyped S1Sf or S3Sf, and
Materials and Methods
Plant materials
were considered self-compatible. The transmission of
In this experiment, 86 progenies from self-pollinated
self-compatibility from 'Tuono', is similar in the two
Touno (S1Sf) almond were prepared in Seed and Plant
progenies 'Ferralise x Tuono' and 'Ferragnès x Tuono.'
Improvement Institute (SPII), Karaj, Iran.
However, in some of the crosses with the female
parent related to Cristomorto, the proportion of selfcompatible individuals was higher than expected (Lopez
et al., 2006). Ortega and Dicenta (2008) studied the
inheritance of self-compatibility in almond. They
observed
that
frequencies
of
self-compatible
descendants were in accordance with the accepted
theory concerning the gametophytic system of Prunus.
Their results confirmed the presence of a common allele
156
Genomic DNA extraction
Total DNA was extracted from young leaves
collected in early spring by the method described by
Murray and Thompson (1980), but modified and
adapted to almond with the difference that 2 μL of βmercaptoethanol
was
added
immediately
after
application of extraction buffer (100 mM Tris–HCl, 1.4
M NaCl, 20 mM EDTA, 2% CTAB, 1% PVP, 0.2%
mercaptoethanol, 0.1% NaHSO3). The purified total
Journal of Nuts 6(2):155-164, 2015
DNA was quantified by gel electrophoresis, and its
DNA polymerase (Cinnagen) and 1 ng of genomic
quality verified
DNA.
by use
of
a
NanoDrop
1000
Spectrophotometer V3.7. DNA samples were stored at PCR amplification
20° C. Three independent extractions were performed
The thermo cycles of the PCR program consisted of
for each sample.
three minutes at 95 °C for primary denaturation, 35
cycles of 94 °C for 1 minute, 53 °C for 1 minute and
PCR reaction
DNA samples with a concentration of 10 ng per μL
then 72 °C for two minutes, followed by 10 min at 72
were prepared for the PCR reaction (Lopez et al., 2006).
°C(Channuntapipat et al., 2003). After PCR, the
Amplification reactions were carried out in 10 μL
products were stored at 4 °C (refrigerator) until
volumes containing: 0.9 mM PCR buffer, 0.6 mM
electrophoresis Profiles of primers used in this study are
MgCl2, 0.9 mM dNTPs, 0.1 mM of each primer
shown in Table 1.
(forward and reverse; Table 1), 0.2 unit of SmarTaq
Table 1. Profile of primers used in this study
Sequence
Primer
Band size (bp)
Visible allele
T annealing
(°C)
CEBASf/AmyC5R
AS1II/AmyC5R
449
Sf
53
1100
S1
60
Marker (locus)
(5′
SFF
AS1II
3′)
GTGCCCTATCTAATTTGTTGAC
TATTTTCAATTTGTGCAACAATGG
Electrophoresis of PCR products
Reference
Channuntapipat
et al. (2003)
Results
Amplified PCR products were separated using 2%
For identification of self-compatible homozygous
agarose gel electrophoresis (Biowittaker Maine, USA)
from heterozygous progenies, 86 seedlings resulting
using 0.5 X Tris–Boric acid–EDTA buffer.
The molecular sizes of the amplification products
from controlled hybridizations of Tuono were studied.
were estimated using a 100-bp DNA ladder (Fermentas).
The results showed that hybrids obtained from crosses
After agarose gel electrophoresis, the gel was stained
made between Tuono had S genotype SfS1 as parents
with ethidium bromide (1 μg/ml) and visualized under
formed bands and these bands considering the especial
UV light using the method of Tamura et al. (2000).
primer indicated that hybrids were 100%
compatible (Table 2) (Figs. 1, 2 and 3).
Table 2. Genotyping of S1, and Sf alleles in progenies
Hybrid
CEBASf/AmyC5R
Second intron
Genotype
TT1
_
1100/450
S1/Sf
TT2
_
1100/450
S1/Sf
TT3
450
450/450
Sf/Sf
TT4
450
450/450
Sf/Sf
TT5
_
1100/450
S1/Sf
TT6
_
1100/450
S1/Sf
TT7
_
1100/450
S1/Sf
TT8
_
1100/450
S1/Sf
TT9
450
450/450
Sf/Sf
TT10
450
450/450
Sf/Sf
TT11
_
1100/450
S1/Sf
157
self-
Journal of Nuts 6(2):155-164, 2015
Table 2. Continued
TT12
_
1100/450
S1/Sf
TT13
450
450/450
Sf/Sf
TT14
_
1100/450
S1/Sf
TT15
_
1100/450
S1/Sf
TT16
_
1100/450
S1/Sf
TT17
450
450/450
Sf/Sf
TT18
_
1100/450
S1/Sf
TT19
_
1100/450
S1/Sf
TT20
_
1100/450
S1/Sf
TT21
_
1100/450
S1/Sf
TT22
_
1100/450
S1/Sf
TT23
_
1100/450
S1/Sf
TT24
450
450/450
Sf/Sf
TT25
450
450/450
S1/Sf
TT26
_
1100/450
S1/Sf
TT27
450
450/450
Sf/Sf
TT28
450
450/450
Sf/Sf
TT29
_
1100/450
S1/Sf
TT30
450
450/450
Sf/Sf
TT31
_
1100/450
S1/Sf
TT32
_
1100/450
S1/Sf
TT33
_
1100/450
S1/Sf
TT34
450
450/450
Sf/Sf
TT35
_
1100/450
S1/Sf
TT36
_
1100/450
S1/Sf
TT37
_
1100/450
S1/Sf
TT38
_
1100/450
S1/Sf
TT39
_
1100/450
S1/Sf
TT40
450
450/450
Sf/Sf
TT41
450
450/450
Sf/Sf
TT42
_
1100/450
S1/Sf
TT43
_
1100/450
S1/Sf
TT44
_
1100/450
S1/Sf
TT45
450
450/450
Sf/Sf
TT46
_
1100/450
S1/Sf
TT47
_
1100/450
S1/Sf
TT48
_
1100/450
S1/Sf
TT49
_
1100/450
S1/Sf
TT50
_
1100/450
S1/Sf
TT51
450
450/450
Sf/Sf
TT52
_
1100/450
S1/Sf
TT53
_
1100/450
S1/Sf
TT54
_
1100/450
S1/Sf
TT55
_
1100/450
S1/Sf
158
Journal of Nuts 6(2):155-164, 2015
Table 2. Continued
TT56
_
1100/450
S1/Sf
TT57
_
1100/450
S1/Sf
TT58
_
1100/450
S1/Sf
TT59
_
1100/450
S1/Sf
TT60
_
1100/450
S1/Sf
TT61
_
1100/450
S1/Sf
TT62
450
450/450
Sf/Sf
TT63
450
450/450
Sf/Sf
TT64
_
1100/450
S1/Sf
TT65
_
1100/450
S1/Sf
TT66
_
1100/450
S1/ Sf
TT67
_
1100/450
S1/Sf
TT68
_
1100/450
S1/Sf
TT69
_
1100/450
S1/ Sf
TT70
_
1100/450
S1/Sf
TT71
450
450/450
Sf/ Sf
TT72
_
1100/450
S1/Sf
TT73
_
1100/450
Sf/S1
TT74
_
1100/450
S1/Sf
TT75
_
1100/450
S1/Sf
TT76
_
1100/450
Sf/ Sf
TT77
450
450/450
Sf/Sf
TT78
_
1100/450
S1/Sf
TT79
_
1100/450
S1/Sf
TT80
_
1100/450
S1/Sf
TT81
450
450/450
Sf/Sf
TT82
_
1100/450
S1/Sf
TT83
450
450/450
Sf/Sf
TT84
_
1100/450
Sf/S1
TT85
_
1100/450
S1/Sf
TT86
_
1100/450
Sf/S1
159
Journal of Nuts 6(2):155-164, 2015
a
100
100
Frequncy(%)
Frequency (%)
8080
6060
b
b
b
4040
2020
00
S1
S1Sf
S1
Sf
S1Sf
A llele
SfSf
Sf
SfSf
Allele
Fig. 1. Frequency of S and F alleles amplified in almond hybrids
M
50bp
59
60
61 62
63
64
65
66
67
68
70
71 72
73
74
75
76 77
78` 79
80
81 82
83
84 85
86
N.C;
1000
500
200
5
Fig.2. Bands of S and F alleles almond progenies using CEBASf and AmyC5R
160
Journal of Nuts 6(2):155-164, 2015
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
25
M
50bp
1100
550
220
M
50bp
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
6
M
50b
p
61
62
63
64
65
66
67
68
70
71
72
73
74
75
76
77
78
79
80
81
82 83
84
85
86
N.C;
1000
1100
500
550
200
220
8
Fig.3. Bands of S1 allele of almond hybrids using AS1 and AmyC5R
161
60
Journal of Nuts 6(2):155-164, 2015
Discussion
progenies in this study can be used in almond
As indicated in Figs. 2 and 3 and Table 2,
self-compatible
breeding programs in future. Thus the identification and
progenies are completely segregated as also reported by
screening for homozygous self-compatibility in almond
Alonso and Socias i Company (2005b). In addition, the
represents another step towards creating an almond
frequency of Sf and S1 alleles were 100 and 50%,
monoculture for use in future research programs.
homozygous
respectively.
and
heterozygous
Similar
results
were
reported
by
Conclusions
Momenpour et al. (2011) who identified self-compatible
Sf alleles in 48almond hybrids using the PCR technique.
The screening of 86 almond offspring of self-
Also, it is interesting to note that the heritability of self-
pollinated Touno by PCR (specific CEBASf and AS1
compatible alleles observed here is consistent with
primers) indicated that the frequencies of Sf, and S1 were
Mendel’s First Law, since the proportion of the self-
100% and 50%, respectively, in progeny. In future
compatible hybrids to total hybrids is 1: 2 (Fig. 1 and
studies selected hybrids can be used in almond breeding
Table 2). If, in the female parent, there is an S1 allele,
programs particularly those aimed at development
all offspring will be the self-compatible, because pollen
monoculture almond orchards.
of the S1 allele of the male parent is unable to growth in
Acknowledgments
to the style tissue and the zygote will not formed; thus
only pollen with Sf alleles is able to penetrate into the
The authors gratefully acknowledge the financial
style and fertilize the ovule, so all offspring will have
assistance provided by the Seed and Plant Improvement
(SfSx, S1Sf) S genotype (Sx= uncertain). These results
Institute (SPII). We would like to thank Mariam Farsi
are consistent with Martin-Gomez et al. (2003),
for expert and helpful comments regarding the PCR
Chanuntapipat et al. (2003), Alonso and Socias i
analysis.
Company (2005a) and Kamali et al. ( 2009). In last part,
it can be said PCR method was used in this study for
identifying
and
screening
homozygous
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