Abstract
Seed orchards with high hereditary qualities and the improvement studies used are of great importance. This study was carried out on individuals in a Boyabat grafted black pine seed orchard, Sinop. The morphological and micromorphological measurements of the characteristics were performed on needle samples taken from individuals, and the genetic diversity was determined on a clonal basis. According to the analysis of variance applied to the data obtained from the measurements and the morphological and micromorphological characters of the clones, it was determined that there was a significant difference among the clones at the P<0.001 confidence level. In this context, according to Duncan’s Range test, the creation of a large number of groups is an indicator of it. The highest heritability rates were obtained in needle diameter, stipule diameter, number of the dorsal stoma, and needle length characteristics.
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Clonal Variation Based on Some Morphological and Micromorphological Characteristics in the Boyabat (Sinop/Turkey) Black Pine (Pinus nigra subsp. pallasiana (Lamb.) Holmboe) Seed Orchard
Nurcan Yigit,a Ayşe Öztürk,a Hakan Sevik,b Halil Barış Özel,c,* Fathi Elmabruk Ramadan Kshkush,a and Berkant Işık c
Seed orchards with high hereditary qualities and the improvement studies used are of great importance. This study was carried out on individuals in a Boyabat grafted black pine seed orchard, Sinop. The morphological and micromorphological measurements of the characteristics were performed on needle samples taken from individuals, and the genetic diversity was determined on a clonal basis. According to the analysis of variance applied to the data obtained from the measurements and the morphological and micromorphological characters of the clones, it was determined that there was a significant difference among the clones at the P<0.001 confidence level. In this context, according to Duncan’s Range test, the creation of a large number of groups is an indicator of it. The highest heritability rates were obtained in needle diameter, stipule diameter, number of the dorsal stoma, and needle length characteristics.
DOI: 10.15376/biores.18.3.4850-4865
Keywords: Black pine; Boyabat; Seed orchard; Morphology; Clones
Contact information: a: Kastamonu University, Faculty of Forestry, Department of Forest Engineering, Kuzeykent, Kastamonu-TURKEY; b: Kastamonu University, Faculty of Engineering and Architecture, Department of Environmental Engineering, Kuzeykent, Kastamonu-TURKEY; c: Bartın University, Faculty of Forestry, Bartın-TURKEY; *Corresponding author: halilbarisozel@gmail.com
INTRODUCTION
Climate types, quite different from each other, prevail across Turkey, and accordingly, there is a wide variety of species in forested areas (Atalay and Efe 2015). However, they enable us to study domestic and foreign species together in afforestation studies. Thus, the opportunities to establish healthy forest stands are increasing in terms of both quality and quantity. In afforestation studies, it is necessary to select appropriate species of the appropriate origin, to follow the principles of improvement studies, and to pay attention to afforestation techniques. For this purpose, the selection of seed sources with high hereditary qualities and the improvement studies used are of great importance (Wu et al. 2015; Kaviriri et al. 2020; Weng et al. 2020).
Improved seeds can increase wood production by up to 40% (Üçler and Turna 2005; Yahyaoğlu and Ölmez 2005). Seeds of a certain origin ensure afforestation and provide economic and ecological benefits (Wu et al. 2015). The most important purpose of tree breeding is to promote ecological and economic benefits (Dyjakon 2019).
Black pine (Pinus nigra Arnold.) is the most important species which can be spread to the steppe regions in Türkiye. Away from its native areas Black pine is planted for its timber production purposes (Topacoglu et al. 2016). Black pine, which is one of the dominant species in the forest assets of Türkiye, is a primary forest tree species that has a very wide distribution area starting from South Europe up to Türkiye. It can be argued that black pine is a typical south European forest tree species that is ecologically and economically important in the abovementioned distribution area (Gülsoy and Cinar 2019).
Depending on changing environmental conditions, it is almost impossible to predict what kinds of threats forests will face in the future. Ensuring the continuity of the genetic diversity of species will make the presence of individuals carrying genes that will be needed in the upcoming years possible (Sevik et al. 2013).
It is preferable for improvement studies that the genetic diversity of the selected populations is as high as possible. It is easier to find proper improvement materials, and the chance of success is higher with populations having a broad genetic base (Velioğlu et al. 2002; Lindgren et al. 2008). High intraspecific genetic diversity is a guarantee of adaptation to changing environmental conditions. Genetic diversity determines the adaptation potential of a species and is an important part of ecosystem stability. Therefore, the conservation of genetic diversity is essential to maintain adaptability. Genetic diversity is a raw material that will be shaped in improvement studies and through which results can be obtained accordingly. High genetic diversity evenly increases the chance of genetic staff to choose genotypes and populations appropriate for their study objectives (Şevik 2012). Seed orchards are one of the most important seed sources in terms of creating a connection between present and future, such as gene conservation areas, and future forest plantations (Bilir and Temiraga 2012).
Seed orchards are areas where seeds are produced in large quantities to obtain the highest genetic gain as cheaply and fast as possible (Wu et al. 2015). According to another definition, they are plantations that are operated for the frequent, abundant, and easy harvesting of forest tree seeds, with use of selected clones or fertilizers, and where pollen flow from isolated or external sources can be blocked or reduced (Zobel and Talbert 1984). They are also shown as the most appropriate way to put the genetic gain obtained from tree breeding studies into practice (Tulukçu et al. 2002).
Genetic diversity can be determined by physiological and morphological characteristics or molecular markers (Suangtho et al. 1999). To date, genetic variation studies have been generally initiated based on morphological characteristics, and after obtaining sufficient data, detailed information has been reached through isoenzyme and DNA studies. However, morphological characteristics were mainly examined in the studies carried out, and the number of studies carried out based on anatomical characteristics has remained quite limited (Donnelly et al. 2016). Studies on needle sizes and anatomical characteristics of conifers showed significant differences between and within populations (Bobowicz and Korczyk 1994; Urbaniak et al. 2003; Androsiuk and Urbaniak 2006). Many studies have been carried out on clonal seed orchards in terms of the morphological characteristics of seeds and cones (Deligöz and Gezer 2005; Çılgın et al. 2007; Hauke-Kowalska et al. 2019; Kaviriri et al. 2020; Weng et al. 2020).
Variation among clones and within clones is an important factor in terms of the seed production (Prescher et al. 2007). Many studies have been carried out on the variations of the productivity of forest tree species (Kang and Lindgren 1998; Benowicz and El-Kassaby 1999; Kang 2001; Bilir et al. 2002; Sengün and Semerci 2002; Bilir et al. 2004; Lindgren et al. 2009).
The study was carried out on grafted black pine individuals in the Boyabat seed orchard, Sinop. The Boyabat Pinus nigra seed orchard, Sinop, was established by taking seed stands of Kastamonu origin. Clones were planted by grafting to seedlings at a distance of 8 × 8 m. The seed orchard is 10.9 Ha in size and located at an altitude of 450 m. This study determined the structuring of adaptive genetic diversity on a clonal basis by analyzing the morphological and micromorphological characteristics of needle samples taken from individuals. For this purpose, the characters measured were analyzed using the SPSS package program, and it was attempted to determine the structuring of adaptive genetic diversity on a clonal basis.
EXPERIMENTAL
The study was carried out in the Boyabat black pine seed orchard, Sinop. The black pine seed orchard affiliated to Sinop Provincial Directorate was planted by the Forest Trees and Seed Improvement Institute in 1995 with 30 clones. From the administrative aspect, the seed orchard is within the boundaries of Kastamonu Regional Directorate of Forestry, Boyabat Forest Management Directorate, Bürnük Forest Sub-district Directorate, and has a size of 10.1 ha (Fig. 1).
Within the scope of the study, genetic variation attributes in the seed orchards were determined with the help of some morphological and anatomical characteristics. Morphological characteristics have been used to determine genetic variations in many studies carried out to date (Sengün and Semerci 2002; Sevik et al. 2013). However, the number of studies in which anatomical characteristics have been used to determine genetic variations is very limited. However, it is known that all phenotypic characters, including anatomical characters, are formed under the mutual interaction of genetic structure and environmental conditions (Yayla et al. 2022; Kuzmina et al. 2023; Cobanoglu et al. 2023). The most dominant factors affecting phenotypic characters are climatic (Koç 2022; Dogan et al. 2023) and edaphic (Cetin et al. 2022; Key et al. 2022) factors. It can be accepted that in seed orchards where climatic and edaphic factors are relatively homogeneous, anatomical characters, like other phenotypic characters, are shaped largely depending on the genetic structure, since they are established in a limited and nearly flat area. In fact, it is accepted that anatomical characters are less affected by environmental conditions and therefore reflect genetic structure more clearly (Yigit 2016; Yigit et al. 2021). Therefore, anatomical characters are important instruments that can be used especially in genetic variation studies, but the number of studies on this subject is negligible.
Fig. 1. Location of the Boyabat black pine seed orchard on the map
Within the scope of the study, needle samples were collected in December, except for the vegetation season. Needle samples were collected from a total of 90 trees, three ramets from each of 30 clones, and last year’s needles from the same direction, labeled, airtight packed, and brought to the laboratory.
Morphological characteristics, such as needle length (NL) (cm), needle width (NW) (mm), needle diameter (ND) (mm), stipule diameter (SD) (mm), number of the dorsal stoma (NDS), number of dorsal stoma channels (NDSC), number of the ventral stoma (NVS), and number of ventral stoma channels (NVSC), were determined on the needle samples collected. Furthermore, ash determination (A) was performed as follows: an empty crucible and cover were dried for 15 min on a heater or in an indirectly heated furnace at approximately 600 °C. The porcelain crucibles were allowed to wait in the desiccator for 45 min and weighed with a precision of 0.1 mg. The sample was put in the crucible. The cover removed and the crucible and its contents were put in an indirectly heated furnace and burned until all carbon was removed. Initially, temperature was gradually increased to avoid volatilization. The crucible was allowed to wait for 3 h at 575 ± 25 °C in the furnace. At the end of this period, the sample in the crucible should be completely bleached, or the particles should have been lost. Ash was calculated as follows,
Ash% = (A*100)/B
where A is the ash weight, and B is the complete dry sample weight (g).
Anatomical characteristics, such as needle wet weight (WW), the weight values of needles taken when they were fresh (g), needle dry weight (DW), and oven-dried weights of samples (at a precision of 0.001 g), were determined.
The data obtained were analyzed using the SPSS 20.0 statistical package program. The analysis of variance was applied to clones in terms of the characters measured, and Duncan’s test was applied to the data in case of statistically significant (P ≤ 0.05) differences as a result of the analysis. As a result of Duncan’s test, new homogeneous groups were determined (Kalıpsız 1994; Ercan 1995).
Broad sense heritability values were estimated both on individual tree basis (H1) and clone mean basis (H2) as the ratio of total genetic variance (s2c) to total phenotypic variance (s2c + s2E) for H1 and to (s2c + s2E/n) for H2 (n = graft number). Cloning effect variance biases heritability values, but the magnitude is negligible and can be ignored. In the present study, heritability components were estimated as E= error mean square and 2c = (clone mean square-error mean square/ number of grafts per clone). This formula has been used in different articles (Sevik and Topacoglu 2015).
RESULTS AND DISCUSSION
As a result of the measurements and calculations performed on the samples taken in the Boyabat black pine clone orchard, A, WW, DW, NL, NW, ND, SD, NDS, NDSC, NVS, and NVSC values were determined. The study was carried out on 141 trees of 30 different clones, and the analysis of variance was applied to the values obtained as a result of the calculations. The results are presented in Table 1. Significant differences were found in terms of all the morphological characteristics studied at a confidence level of 99.9% both among the clones and within the clones. Duncan’s test was applied for each anatomical characteristics to determine how the clones were grouped within themselves, and the test results are presented in Table 2.
According to Duncan’s test results of the A character, clone 81 (1.839) formed a class alone, and clone 87 had the highest value (1.361) and was included only in the last homogeneous group according to the ash content of the needle. According to the wet weight values of the needle, clone 95 with the lowest value (1.833) was included only in the first homogeneous group, and clone 82 with the highest value (2.11) was included only in the last homogeneous group. According to the results of Duncan’s test applied to the dry weight values of the needle, Clone 95 was included only in the first homogeneous group (0.817) according to the dry weight values of the needle and that clones 1 and 82 (2.003 and 2.087) were included only in the fifth and last homogeneous group (Table 2).
In terms of the NL morphological character, the clones formed 16 different classes among themselves. Clone 85 was included in the first homogeneous group with a needle length value of 10.5 cm and clone 93 was included in the last homogeneous group with a needle length value of 12.6 cm. In terms of the ND morphological character, it is observed that three different groups were formed. Clone 88 was included in the first homogeneous group, clone 93 was included in the second homogeneous group, and all other clones were in the 1st class. In terms of the NW character, the test revealed that 15 different groups were formed. When the groups were examined, clone 85 was included in the 1st homogenous group, clones 81 and 85 were included in the 2nd homogenous group, and clone 8 was included in the last homogeneous group alone. In terms of the SD morphological character, clone 85 formed a homogeneous group alone, and clone 81 formed the 2nd homogenous group. Clone 93 was included in the last homogenous group (Table 2).
The clones were grouped in 11 different groups in terms of the NDS character according to Duncan’s test results of micromorphological characteristics. Clone 98 formed the first and single group and clones 95 and 97 formed the second group together. Clone 89 formed the last class alone. When Duncan’s test results were examined in terms of the NDSC character, 7 groups were formed. Clone 82 formed the last class alone and clones 85 and 89 formed a class together. When Duncan’s test results were examined in terms of the NVS micromorphological character, clones 86, 93, 88, 96, and 10 formed a class by themselves. Clone 8 formed the last class alone. When the results of Duncan’s test applied to micromorphological characteristics were examined in terms of the NVSC character, clones 7, 93, and 99 formed a class by themselves and clone 8 formed the last class.
Correlation analysis was performed to determine the relationship between the elements, and the results are presented in Table 3. The studied relationship that was not measured in the correlation analysis was related to the linear part of the relationship between the variables.
Table 1. Analysis of Variance Applied to All Characteristics
P<0.05 (95% confidence level); P<0.01 (99% confidence level); P<0.001 (99.9% confidence level) ns: Not significant
Table 2. Duncan’s Test Results of Ash Content in All Characteristics
The correlation coefficient calculated as a result of the correlation analysis was indicated with r and took values between –1 and +1. The fact that the coefficient was close to +1 indicates that there was a good correlation between the two variables, and the fact that it was close to -1 indicates that there was a good but inverse correlation, in other words, one of the variables increased while the other one decreased. Upon evaluating the results in this respect, it is observed that the level of relationship between some elements was high.
Table 3. Correlation Analysis Results