Effective Clone Number and Contribution to Gene Pool in a Clonal Seed Orchard of Anatolian Black Pine (Pinus nigra Arnold subsp. pallasiana (Lamb.) Holmboe) in Kastamonu-Turkey

Abstract

Aim of study: Clonal seed orchards are really important tools in seed material production. In this study; the effective clone number and clone contribution to gene pool were examined.Area of study: The studied clonal seed orchard was established on 13 ha of area, located at Hanönü (Kastamonu) in Turkey.Material and Methods: In 2008, the existed ramets were firstly counted for determining the ramet number for the clones. After that, all cones on the ramets were counted and CGP (clone contribution to gene pool) was analysed. The effective number of clones (Nc) was determined by the variation coefficient (CV).Main results: There is considerable variation on living ramet numbers among the clones. In the establishment phase, there was also 7 times difference between clone 22 (12 ramets) and clone 2 (85 ramets). This difference both establishment phase and 17 years-old, could stem from epibiot-hipobiot incompatibility and environmental factors.Highlights: The effective number of clones, describes the gene pool of the orchard. However, incorporation of fertility variation among clones with variation in the number of ramets will give a better prediction of it.

Full text

Research Article Kastamonu Uni., Orman Fakültesi Dergisi, 2019, 19(1): 137-142
Kastamonu Univ., Journal of Forestry Faculty
Doi:10.17475/kastorman.543551
137
Effective Clone Number and Contribution to Gene Pool in a Clonal
Seed Orchard of Anatolian Black Pine (Pinus nigra Arnold subsp.
pallasiana (Lamb.) Holmboe) in Kastamonu-Turkey
Ahmet SIVACIOĞLU1*, Osman TOPAÇOĞLU1
1Kastamonu University, Faculty of Forestry, Department of Forest Engineering, KASTAMONU
*Corresponding author: asivacioglu@kastamonu.edu.tr
Received Date: 03.02.2019 Accepted Date: 11.03.2019
Abstract
Aim of study: Clonal seed orchards are really important tools in seed material production. In this
study; the effective clone number and clone contribution to gene pool were examined.
Area of study: The studied clonal seed orchard was established on 13 ha of area, located at Hanönü
(Kastamonu) in Turkey.
Material and Methods: In 2008, the existed ramets were firstly counted for determining the ramet
number for the clones. After that, all cones on the ramets were counted and CGP (clone contribution to
gene pool) was analysed. The effective number of clones (Nc) was determined by the variation coefficient
(CV).
Main results: There is considerable variation on living ramet numbers among the clones. In the
establishment phase, there was also 7 times difference between clone 22 (12 ramets) and clone 2 (85
ramets). This difference both establishment phase and 17 years-old, could stem from epibiot-hipobiot
incompatibility and environmental factors.
Highlights: The effective number of clones, describes the gene pool of the orchard. However,
incorporation of fertility variation among clones with variation in the number of ramets will give a better
prediction of it.
Keywords:
Pinus nigra,
ramet variation, effective clone number, Kastamonu.
Anadolu Karaçamı (Pinus nigra Arnold subsp. pallasiana (Lamb.)
Holmboe) (Kastamonu-Turkey) Klonal Tohum Bahçesinde Etkili Klon
Sayısı ve Gen Havuzuna Klon Katkısı
Öz
Çalışmanın amacı: Klonal tohum bahçeleri nitelikli tohum üretiminde önemli bir yer tutmaktadır. Bu
çalışmada etkili klon sayısı ve klonların gen havuzuna katkıları incelenmiştir.
Çalışma alanı: İncelenen tohum bahçesi 13 ha alanda, Hanönü (Kastamonu), Türkiye’de tesis
edilmiştir.
Materyal ve Yöntem: 2008 yılında öncelikle klonların mevcut ramet sayılarını belirlemek için bütün
rametler sayılmıştır. Daha sonra toplam kozalak sayısı belirlenerek CGP (klonların gen havuzuna
katkıları) analizi edilmiştir. Etkili klon sayıları (Nc), rametlerin varyasyon katsayısına (CV %) göre
belirlenmiştir.
Sonuçlar: Yaşayan klonların ramet sayıları arasında büyük fark bulunmaktadır. Tohum bahçesinin
kuruluş aşamasında da klonlara ait ramet sayıları arasında 7 kat fark, klon 22 (12 ramet) ve klon 2 (85
ramet) bulunmaktadır. Kuruluş aşamasında ve 17 yaşında ramet sayıları arasında görülen bu büyük fark
aşı uyuşmazlığından ve çevresel faktörlerden kaynaklanmaktadır.
Önemli Vurgular: Etkili klon sayısı tohum bahçesinin gen havuzunu tanımlamaktadır. Bunun
yanında, tohum ürününün genetik çeşitliliği ile ilgilendiğimiz zaman, ramet sayılarının farklılığından
kaynaklanan döllenme çeşitliliği daha iyi bir öngörü sağlamaktadır.
Anahtar Kelimeler:
Pinus nigra
, ramet varyasyonu, etkili klon sayısı, Kastamonu.
Citation (Atıf): Sivacioglu, A. & Topacoglu, O. (2019). Effective clone
number and contribution to gene pool in a clonal seed orchard of
Anatolian Black Pine (Pinus nigra Arnold subsp. pallasiana (Lamb.)
Holmboe) in Kastamonu-Turkey. Kastamonu University Journal of
Forestry Faculty, 19 (1), 137-147.
This work is licensed under a Creative Commons
Attribution-NonCommercial 4.0 International
License.

Kastamonu Uni., Orman Fakültesi Dergisi, 2019, 19(1): 137-142 Sivacioglu and Topacoglu
Kastamonu Univ., Journal of Forestry Faculty
138
Introduction
Anatolian black pine is one of the
commercially important tree species in
Turkey. Also, the area of this species spreads
on 4.2 million ha, of which almost 1.8
million ha is degraded forests (Anonymous,
2006). Because of high rate of unproductive
forest lands, big amount of seed material is
needed for restoration of unproductive lands.
Moreover, this pine species seems to be one
of the most important species which can be
widely used in arid/semi-arid regions in
Anatolia. Recently, the semi-arid steppe
lands considered as potential afforestation
areas by Turkish Foresters. Thus, the
importance of Anatolian black pine is
increasing continuously. In the present case,
the seed material is supplied from 55 (464
ha) of seed orchards and 71 (9087.5 ha) of
seed stands in this species (Anonymous,
2019). Mostly, these seed orchards are rather
young, because they were set after the year
of 1990 (Sıvacıoğlu and Ayan, 2010).
The importance of seed orchard are
appreciated as production populations where
genetic gain from tree breeding is transferred
into practices and to commercial forest crops.
The actual seed orchards are set up as to
phenotypic selection of plus individuals from
seed stands in Turkey. In the time of
establishment of clonal seed orchards,
selected genotypes (plus trees) are replicated
with grafting. After definite period (1 or 2
years) these grafts (ramets), are planted as to
a certain design for avoiding self-fertilization
and consequent seed production (Kang et al.,
2001; Tunçtaner, 2007). Clones in new
established clonal seed orchards are in equal
proportions (Koski, 1980). The clone
number (census number), in an seed orchard,
is thus currently used for regulatory purposes
and is regarded as essential for decisions on
whether forest genetic material is suitable to
use. But, the census number has no meaning
in terms of gene diversity in case of large
variation in ramet numbers per clone and in
fertility among genotypes (Tunçtaner, 2007).
In the discussions about inbreeding and
genetic diversity in clonal seed orchards
focusing on the clone numbers (Koski, 1980;
Lindgren, 1974; Moran, Bell & Matheson,
1980), it is often assumed that all clones are
represented by close-to-equal numbers of
fertile ramets (Jonsson, Ekberg & Eriksson,
1976; Hodge & White, 1993; Xie, Woods, &
Stoehr, 1994; Kang & Lindgren, 1998).
However, it is generally acknowledged that
clones are quite often represented by
differing numbers of ramets in actual clonal
seed orchards (Schoen, Denti & Stewart,
1986; Muona and Harju, 1989; Bilir and
Ayan, 2005). Together with variation in
fertility, this variability in ramet number will
be reason for unequal gamete contributions
by clones in the orchards. In case of
unrelated and non-inbred clones, the
effective number of clones in an orchard will
be equal to the status number of the seed
orchard (Lindgren and Kang, 1997; Lindgren
and Mullin, 1998). Further, it can also be
regarded as the status number of the seed
crop from an ideal seed orchard where clones
are non-inbred and not related, and where
gamete contributions are proportional to the
ramet number (Lindgren and Mullin, 1998).
In this study, to determine the effective
clone number based on clone and ramet
number, and to evaluate of ramet
variation on gene diversity in a clonal
seed orchard of Anatolian Black Pine
was aimed. Moreover, the findings were
evaluated to shed light on future and present
management of clonal seed orchards.
Material and Method
The material seed orchard was established
by Research Institute of Forest Tree Seeds
and Tree Breeding on 13 ha of area, located
at Hanönü (Kastamonu) Turkey in 1993 with
2039 grafts of 30 clones, taken out from
chosen trees in Kastamonu-Karadere seed
stand. Grafts were 1 year-old at the time of
set up and planting space was 8 m x 8 m.
First cones were harvested in 2003. In
2008, the existed ramets were counted firstly
for determining the ramet number for the
clones. Later, all cones on the ramets were
counted and CGP (clone contribution to gene
pool) was analysed.
The effective clone number (Nc) for the
studied orchard was determined by using the
variation coefficient (CV) for the ramet
numbers representing each clones. The Nc
value is determined as Nc=N (census
number) /(CV2+1). In case of unrelated, non-
inbred parents, parental fertility in

Kastamonu Uni., Orman Fakültesi Dergisi, 2019, 19(1): 137-142 Sivacioglu and Topacoglu
Kastamonu Univ., Journal of Forestry Faculty
139
commensurate with number of ramets and
moreover inclusion of all parents,
effective clone (NC) number turn into
orchard status number (NS) (Kang et al.
2001, Lindgren & Mullin 1998).
There is a relation between gene diversity
(GD) with effective clone numbers as,
GD=1-0.5/NC (Lindgren and Kang, 1997) or
with status number (NS) as, GD = 1- 1/
(2*NS). Moreover, this relation can be
stated also with the relative effective clone
number (Nr) and clone numbers (N) as,
(GD=1-0.5/N*Nr). The highest level of gene
diversity of seed material for a certain clone
number is acquired whenever all parents join
to the gene pool of the seed orchard at an
equal level (Kang et al., 2001). Thus situated
variation coefficient (CV) among clones for
ramet numbers take on an important task in
seed crop gene diversity. To confront the
census number (N) and the clone effective
number (Nc) in clonal seed orchard, mostly
their relation as, Nr = NC /N was used (Kang
et al., 2001; Kang and Lindgren, 1998; Bilir
and Ayan, 2005; Lindgren and Kang, 1997).
Analyses of variance were performed for
cone numbers. To data of cone number,
one-way variance analysis applied. Thus,
normality/homogeneity tests and logarithmic
transformation were done. Variation
coefficient within the clones (CVC) and
grafts (CVG) in percent was determined as
the ratio of standard deviation of the
clones/grafts to arithmetic means.
Results
There is large differences for living
number of ramets among clones. For
instance; in 2008, there is 8 times differences
between lowest (clone 22, 8 ramets) and
highest (clone 2, 66 ramets) number of
ramets per clone. In the establishment phase,
there was also 7 times difference between
clone 22 (12 ramets) and clone 2 (85 ramets)
(Table 1).
This difference both establishment phase
and in 17 years-old, could stem from epibiot-
hipobiot incompatibility and environmental
factors. In 17 years period, the highest MP
(mortality percentage) observed for clone 29
(50 %), whereas the lowest for clone 6 (18.18
%) (Table 1).
All clones combined, the orchard had a
mean of 122.4 cones with range among the
clones from 32.8 (clone 28) to 189.8 (clone
13). The CVG values varied from 66.8 to
129.9 while the variation coefficients among
the clones (CVC) is 31.1. The bigger CVG
values than CVC indicates the bigger
variation within the clones than among the
clones in the orchard for cone number. The
analysis of variance showed that there was
significant differences among clones at 0.001
probability level for CGP (Table 1). The
CGP varied 0.83-5.46% among the clones.
The top of 5 clones (20, 21, 10, 15, 13) had
24.82 % of all CGP (Table 1).
The CV values varied from 22.3 % in
1993, to 27.1 % in 2008. As to the data, there
is a negative significant (p<0.001) relation
(r=-0.998) between CV of ramet number and
Nc (Nc=-0.14CV+31.82). Thus, Nr values
varied from 0.95 in 1993 to 0.93 in 2008
(Table 2). In 1993, each clone represent with
a mean of 69.6 grafts, whereas this value
decrease to 48.0 in 2008. Total graft number
varied 2089 to 1441, from 1993 to 2008
(Table 2).
Discussion
In Turkey, the existed clonal seed
orchards were commonly established using
ramets of phenotypically selected plus trees,
without information on breeding value
(Tunçtaner, 2007; Hodge & White, 1993). It
is often considered preferable to use an equal
number of ramets per clone whenever
possible, and there was no reason not to see
equal ramet representation as an aim in the
first cycle of seed orchards (Kang et al.,
2001). In the process of orchard
establishment, however, unintentional
variation in the number of ramets for selected
clones occurs. For example, in the studied
orchard, ramet numbers varied from 12 to 85
in the establishment phase. One of the major
reason of this variation may be graft
availability. Once planted, grafts may die for
different reasons such as unfavourable site
growing conditions, graft incompatibility,
different kinds of biotic or abiotic injuries. In
the studied orchard, mostly this variation
originated from unfavourable site conditions.

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Table 1. Ramet numbers (RN), total cone number (TCN), contribution of clones to gene pool of
orchard (CGP) and descriptives for studied orchard.
Clone
number
RN
1993
RN
2008
MP
TCN
CGP4
%
Mean
SE
Min-Max
SDG
CVG%
1
81
62
23.46
7240
4.26
bcde2
1116.8
11.9
1-340
93.9
80.4
2
85
66
22.35
7351
4.32
bcde
111.4
15.8
0-680
128.7
115.5
3
79
59
25.32
5771
3.39
efgh
97.8
16.1
0-720
123.4
126.2
4
80
51
36.25
5785
3.40
efgh
113.4
11.1
0-300
79.3
69.9
5
77
56
27.27
6277
3.69
defg
112.1
12.5
0-348
93.6
83.5
6
77
63
18.18
7321
4.31
bcde
116.2
10.5
0-440
83.6
71.9
7
75
53
29.33
5298
3.12
fghi
99.9
13.2
3-480
96.5
96.6
8
45
30
33.33
4733
2.78
ghij
157.7
24.4
0-520
133.6
84.7
9
73
56
23.29
7127
4.19
cde
127.2
15.2
0-520
114
89.6
10
80
60
25.00
8153
4.80
abc
135.8
13.1
0-428
101.2
74.5
11
58
47
18.97
5085
2.99
fghij
108.2
12.1
3-292
83.3
77.0
12
36
28
22.22
4705
2.77
ghij
168
21.2
15-414
112.2
66.8
13
73
49
32.88
9288
5.46
a
189.5
18.3
0-480
128.5
67.8
14
75
47
37.33
5158
3.03
fghij
109.7
14.4
0-396
99.2
90.4
15
73
49
32.88
8873
5.22
ab
181.1
18.3
0-560
128.3
70.8
16
59
28
52.54
4539
2.67
ghij
162.1
21.9
11-560
116.2
71.7
17
75
41
45.33
5357
3.15
fghi
130.6
16.1
6-464
103.5
79.2
18
77
54
29.87
4987
2.93
ghij
92.3
9.9
0-260
73.1
79.2
19
69
45
34.78
3973
2.34
ij
88.3
10.4
0-260
69.9
79.2
20
80
56
30.00
7810
4.59
abcd
139.4
15.4
0-460
115.4
82.8
21
78
58
25.64
8077
4.75
abc
139.3
15.3
0-460
116.9
83.9
22
12
8
33.33
1410
0.83
l
176.2
56.7
13-408
160.5
91.1
23
62
38
38.71
7214
4.24
bcde
189.8
22.8
0-620
141.1
74.3
24
74
41
44.59
3542
2.08
jk
86.4
17.5
0-540
112.2
129.9
25
75
54
28.00
6798
4.00
cdef
125.9
15.1
0-620
110.6
87.8
26
76
50
34.21
2325
1.37
kl
46.5
6.2
0-132
43.9
94.4
27
77
57
25.97
6246
3.67
defg
109.6
11
0-340
83.3
76.0
28
79
54
31.65
1773
1.04
l
32.8
5.2
0-152
38.7
118.0
29
54
27
50.00
3479
2.05
jk
128.8
21.8
0-380
113.6
88.2
30
75
54
28.00
4305
2.53
hij
79.7
7.3
0-196
53.6
67.3
Mean
69.6
48.0
5666.6
3.33
122.4
7.0
32.8-189.8
38.1
31.1
F value
314.578***
1Values are the means of all grafts.
2For each trait, mean values with the same letter are not significantly different at P < 0.05 level;
3 ***: significant at P < 0.001 and the values are transformed.
4 RN=Ramet number, MP=Mortality percentage TCN=Total cone number CGP= contribution percentage of clone to
gene pool SE=standard error SDG= Standard deviation among the grafts CVG =Variation coefficients among the grafts
Table 2. Clone and graft number, effective clone number (Nc), relative effective number of
clones (Nr), variation coefficients of grafts (CV%) , Gene diversity (GD) of the studied orchard.
Year
Clone
number
Graft number
Nc
Nr
CV%
Average
Total
Min.
Max.
1993
30
69.6
2089
12
85
28.6
0.95
22.3
2008
30
48.0
1441
8
66
27.9
0.93
27.1

Kastamonu Uni., Orman Fakültesi Dergisi, 2019, 19(1): 137-142 Sivacioglu and Topacoglu
Kastamonu Univ., Journal of Forestry Faculty
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Also, the genetic differences among
clones may contribute to this mortality
causing variation in the number of living
ramets for individual clones in the studied
orchard. As seen on Table 1, the mortality
percentage varied from 18.18 to 52.54 among
the clones. Also, the ramet number variation
may arise also from non-genetic reasons such
as different grafters, root-stock quality,
storage facilities. If grafts die soon after
planting, they may be replaced by reserve
ramets that may represent clones where
initial grafting was successful or which was
not used for the intended seed orchard.
The effective number of clones (Nc),
based on the variation in the number of
ramets among clones, describes the gene
pool of the orchard. However, when we are
interested in the gene diversity of the seed
orchard crop, incorporation of fertility
variation among clones with variation in the
number of ramets will give a better
prediction of it. Also, Nc will be more
informative concerning gene diversity of the
seed crop than is the census number (N). It
was developed status effective number (Ns)
in connection with fertility variation in seed
orchards. Loss in gene diversity is inversely
proportional to the effective number of
clones (Kang & Lindgren, 1998). On
average, the effective number of clones was
calculated to be 28.6 in 1993, 27.9 in 2008.
The census number (30) of clones differs
from the effective number of clones in the
studied clonal seed orchard.
There is no big difference on relative
effective number of clones (Nr) between
1993 and 2008 (0.95 and 0.93). But, what is
important here is the reduction of the
contribution of some clones to the gene pool.
It is beneficial and more desirable in
future orchards than at present to
intentionally use an unequal number of
ramets per clone. So, the clones that
contribute most to breeding value of the seed
orchard are permitted to contribute more to
the diversity loss.
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