New Forests (2006) 31:409–416 DOI 10.1007/s11056-005-0876-x
Springer 2006
-1
Mating system and pollen contamination in a Pinus brutia seed orchard NURAY KAYA1,*, KANI ISIK1 and W.T. ADAMS2 1
Department of Biology, Faculty of Arts and Sciences, Akdeniz University, 07058 Antalya, Turkey; Department of Forest Science, Oregon State University, Corvallis, OR 97331, USA; *Author for correspondence (e-mail:
[email protected]; phone: +90-242-310-23-66; fax: +90-242-227-8911) 2
Received 10 October 2003; accepted in revised form 5 July 2005
Key words: Gene flow, Isozymes, Outcrossing, Pine, Selfing Abstract. Mating system parameters and pollen contamination were estimated in an 11-year-old Pinus brutia Ten. clonal seed orchard, located in Antalya, Turkey, with the aid of isozymes. Isozyme analysis was performed on both maternal (megagametophyte) and embryo tissues of seeds collected from the seed orchard and the surrounding natural stand. Fourteen loci encoding nine enzyme systems were assayed. Based on a multilocus estimator, the proportion of viable seeds originating from outcrossing (tˆm) in the seed orchard was found to be 0.947; the remainder (0.053) was due to selfing. We estimated that 85.7% of the orchard seed resulted from pollen contamination by trees in surrounding stands. This may be related to the young age and subsequent low pollen production of the orchard. Only 9% [i.e., 1.0 0.857 (contamination) 0.053 (selfing) = 0.090] of the orchard offspring was due to cross-pollination among clones within the orchard. Due to the high level of pollen contamination, it is estimated that genetic gain in this seed crop is less than 57% of that expected if all offspring resulted from random mating among orchard clones (i.e., no contamination).
Introduction Seed orchards are established in order to produce seeds easily, abundantly, and economically from trees with desirable genetic properties (Zobel et al. 1958; El-Kassaby et al. 1989; Di-Giovanni and Kevan 1991). Seed orchards are of vital importance in genetic improvement of forest trees and the short and longterm success of plantations depend on the genetic quality of seeds that orchards produce. The genetic quality of orchard crops is influenced by many factors (Fast et al. 1986; Ladislav and Go¨mory 1995); among them, pollen contamination and selfing need to be particularly avoided. Pollen contamination in wind-pollinated seed orchards decreases genetic gain (Wheeler and Jech 1986). Previous reports have estimated levels of pollen contamination in conifer orchards ranging from 30 to 90% (Wheeler and Jech 1986; Di-Govanni and Kevan 1991; Adams et al. 1997). In addition, inbreeding, particularly selfing, often leads to reduction in seed yields and poor growth of viable seeds (Erickson and Adams 1990; Adams et al. 1992). Information on levels of selfing and pollen contamination is critical to evaluating the quality of
410 seed produced under wind pollination, and the need for applying pollen management techniques (e.g., flower stimulation, supplemental mass pollination) to limit the negative impacts of these factors (Adams and Birkes 1991). Isozyme analysis has been widely used for estimating mating system parameters and pollen contamination in seed orchards of different forest tree species (Smith and Adams 1983; Friedman and Adams 1985; Greenwood and Rucker 1985; Nagasaka and Szmidt 1985; El-Kassaby et al. 1989; Harju and Muona 1989; Adams and Birkes 1991; Adams et al. 1997). These estimation procedures are aided in conifers by the presence of two types of seed tissue which make it possible to genotype individual pollen grains effective in fertilization: the haploid megagametophyte tissue (maternally derived) and the diploid embryo. In this paper, we report the first estimates of pollen contamination and mating system parameters in a Pinus brutia Ten. clonal seed orchard.
Material and methods Seed orchard and seed samples The orchard in this study is located at Asar, Antalya in southern Turkey (3701¢30¢¢ N latitude and 3043¢30¢¢ E longitude, 240 m altitude). Southern and western sides of the orchard are surrounded by natural P. brutia stands, each about 100 m distance from the orchard edge (Kaya 2001). The orchard consists of 28 selected clones originating from a seed stand located 125 km away, between 3706¢20¢¢ latitude and 2907¢30¢¢ longitude, at Cameli-Goldagi, near Denizli in southwestern Turkey. The orchard, which is 11.2 ha in size, was established in 1986 with each clone represented by an average of 60 ramets, and grafts planted at an 8 · 8 m spacing (Kaya 2001). Cones were collected in 1997 from three grafts (ramets) per clone, each distributed widely across in the orchard, when ramets were 11 years old and averaged 5.1 m in height. Cones were also collected from 60 trees distributed widely in the surrounding natural stands. The cones were first dried at room temperature, and then the seeds were extracted, cleaned, and kept separate and labeled by mother tree or ramet. The seeds were stored at 4 C until laboratory analysis.
Electrophoretic methods For the laboratory analyses, the seeds were germinated on moistened Whatman N3 filter paper in petri dishes at 24C. The seeds were dissected and the haploid megagametophyte and embryo tissues homogenized separately in 75 ll of 0.2 M Phosphate buffer (pH 7.5) (Kara et al. 1997). The resulting homogenates
411 were subjected to starch gel electrophoresis (12% starch) using four different gel buffer systems according to slightly modified methods of Conkle et al. (1982) described in Kara et al. (1997) and Kaya (2001). In total, nine enzyme systems, encoded by 14 loci, were assayed. These are listed in Table 1 along with their enzyme commission numbers, abbreviations, gel buffers used, and numbers of loci scored. Details on the inheritance of these enzymes are reported by Kara et al. (1997).
Genetic analysis Pollen contamination analysis requires genotypic identification of all clones in the seed orchard. Therefore, we analyzed haploid megagametophyte and embryo tissues of 16 seeds from each ramet. Multilocus allozyme genotypes of all 84 sampled ramets were inferred from the alleles observed in their megagametophytes. With a sample of 16 megagametophytes, the probability (p) of misclassifying a heterozygous maternal genotype at any single locus is less than 3.05 · 105 [p = (1/2)n 1 = (1/2)15]. Pollen contamination in the seed orchard from surrounding populations was estimated using the multilocus estimation procedure of Smith and Adams (1983) and GENFLOW, a computer program written by Adams and Burczyk (1993). For each seed, we determined the genotype of the fertilizing pollen grain by comparing the genotype of the embryo and the megagametophyte. The total seed sample for estimating pollen contamination was approximately 1344 (16 seeds · 3 ramets · 28 clones). We also assayed eight megagametopyhes in seeds from each of the 60 trees sampled in the background population. Thus, we were able to estimate allele frequencies in the background population. Details of the multilocus estimation procedure for pollen contamination are found elsewhere (Smith and Adams 1983; Adams and Birkes 1991); only a brief description follows. The first step was to compare the 11-locus genotype of Table 1. The assayed enzymes, their abbreviations, enzyme commission numbers, gel buffers used, and numbers of loci scored in Pinus brutia. Enzyme’s name
Abbreviation
E.C. No.
Buffera
Numbers of loci scored
Aconitase Alcohol dehydrogenase Glutamate dehydrogenase Glutamate oxaloacetate-transaminase Malate dehydrogenase Menadione reductase 6-Phosphogluconate dehydrogenase Phosphoglucoisomerase Shikimate dehydrogenase
Aco Adh Gdh Got Mdh Mnr 6pgd Pgi Sdh
4.2.1.3 1.1.1.1 1.4.1.2 2.6.1.1 1.1.1.37 1.6.99.2 1.1.1.44 5.3.1.9 1.1.1.25
MC6.1 TBE TC TC MC8.1 TBE MC6.1 MC8.1 MC6.1
1 1 1 3 1 2 2 2 1
a MC6.1 – Morpholine Citrate (pH 6.1); MC8.1 – Morpholine Citrate (pH 8.1); TBE – Tris–Borate– EDTA; TC – Tris–Citrate. Details on the gel buffers are reported in Kara et al. 1997.
412 each pollen gamete in the seed crop sample with gamete genotypes that can be produced by the 28 clones in the orchard. All pollen gametes that could not have been produced in the orchard (i.e., do not match any of the gamete genotypes of the 28 clones) were detected contaminants. The total of detected contaminants is a minimum estimate of contamination. The multilocus genotypes of some gametes from background sources can match those produced by seed orchard clones and, thus they will be undetected. To determine the true proportion of contamination (m), it is necessary to calculate the probability that a contaminant pollen grain has a detectable genotype (detection probability) (d). If the genetic composition of the background pollen gamete gene pool is known, d can be estimated (Smith and Adams 1983). The true proportion of pollen contamination (m) is then estimated as: ^¼ m
b^ d^
where, bˆ = the observed proportion of detected contaminants, and dˆ = the estimated detection probability. To further quantify mating in this orchard, we estimated t, the proportion of viable progeny resulting from random outcrossing (where t = 1 s, and s is the proportion due to selfing). A multilocus estimate of t (tˆm) was calculated using the same pollen gametes (i.e., from 48 seeds per clone) and 11 loci analyzed for pollen contamination, by employing the MLTR computer program written by Ritland (1998) and based on the Maximum Likelihood procedure of Ritland and ElKassaby (1985). When orchard seed results from fertilization by pollen from unimproved trees, predicted genetic gain is one-half of that expected under no contamination (Squillace and Long 1981), and assuming no loss in productivity due to maladaptation. The net overall gain (Gn) is therefore: Gn ¼ G
^ Gm 2
where, G = the gain expected under no contamination.
Results and discussion Allele frequencies in the seed orchard and in the neighboring stand of P. brutia were significantly different (p < 0.01) at six of the 14 loci assayed (Table 2). In addition the neighboring stands had alleles that are absent in the seed orchard (e.g. Aco-2, Got1-2, and Got3-2) (Table 2). This is expected because of both the small number of clonal genotypes in the orchard (28) and geographic origin of the clones which is different from that of the background stands (Sniezko 1981; Squillace and Long 1981).
413 Table 2. Allele frequencies in the P. brutia seed orchard and in the background stands. Locus
Allele
The orchard
Background
v2 (df)a
p
Aco
1 2 1 2 1 1 2 1 2 1 2 1 2 1 1 2 1 2 3 1 2 3 1 1 2 1 2 3
1.000 0.000 0.025 0.975 1.000 1.000 0.000 0.618 0.382 1.000 0.000 0.318 0.682 1.000 0.870 0.130 0.613 0.353 0.034 0.627 0.152 0.221 1.000 0.654 0.346 0.305 0.553 0.142
0.936 0.065 0.032 0.968 1.000 0.984 0.016 0.919 0.081 0.992 0.008 0.226 0.774 1.000 0.911 0.089 0.339 0.661 0.000 0.669 0.097 0.234 1.000 0.566 0.434 0.237 0.407 0.356
106.89 (1)
***
0.12 (1)
ns
0.00 (1) 26.68 (1)
ns ***
23.22 (2)
***
13.33 (1)
***
1.91 (1)
ns
0.00 (1) 0.90 (1)
ns ns
25.16 (2)
***
1.42 (2)
ns
0.00 (1) 2.01 (1)
ns ns
20.49 (2)
***
Adh2 Gdh Got1 Got2 Got3 Mdh1 Mnr1 Mnr2 6pgd2
6pgd3
Pgi1 Pgi2 Sdh1
a
Test of allele frequency differences between the orchard and background stands. ***Significant at the 0.1 percent level; ns – Nonsignificant.
The proportion of observed contaminants (bˆ) in seed orchard offspring was 0.179, the detection probability (dˆ) was 0.209, and the estimated pollen contamination (mˆ) was 0.857. Although this level of pollen contamination is quite high, it falls within the range reported for several earlier seed orchard studies in conifers (Fast et al. 1986; Adams and Burczyk 2000). Squillace and Long (1981), Adams and Birkes (1989), and Greenwood and Rucker (1985) estimated pollen contamination levels of 83.5% in Pinus elliottti, 91% in Pseudotsuga menziesii, and 31–88% in Pinus taeda, respectively. Also, Fast et al. (1986) pointed out that pollen contamination in a P. menziesii seed orchard was 0.44 and 0.89 in two consecutive years. In general, pollen contamination contributes to increased genetic variation in seed orchard crops, but at the expense of decreased genetic gain, and perhaps reduced adaptability (if the orchard clones are adaptively different from background populations, Fast et al. 1986; Wiselogel 1986). Levels of pollen contamination fluctuate over time depending on several factors, including weather conditions prior to and during the flowering period,
414 timing of female cone receptivity of orchard clones relative to orchard and background pollen sources, level of pollen production inside the orchard, and level of pollen production in background stands (Harju and Muona 1989; Pakkanen and Pulkinen 1991). The high pollen contamination observed in this study is most likely due to the relatively young age (11 years since grafting) of the ramets in the P. brutia seed orchard. Trees in the surrounding stands exhibited mixed age structure, with the oldest trees being over 100 years. Surely, the amount of pollen produced by the clones in the orchard was considerably lower than that produced in the nearby natural stands. Wang et al. (1991), Harju and Muona (1989), and Pakkanen and Pulkinen (1991) also concluded that the relatively high amounts of pollen contamination observed in their orchards were mainly due to the low production of pollen by the orchard trees. The estimated proportion of outcrossed seed in the orchard crop (tˆm) was very high (0.947), which is typical for most conifer species (Omi and Adams 1985; Ritland and El-Kassaby 1985; El-Kassaby et al. 1989; Harju and Muona 1989; Ladislav et al. 1993), and lies within the range reported for several other pines (El-Kassaby et al. 1987; El-Kassaby et al. 1989; Krutovskii et al. 1995; Burczyk et al. 1996; Burczyk et al. 1997). A high outcrossing rate (0.947) means a low proportion of viable selfed offspring. The actual level of self-fertilization, however, may be much greater because most selfs result in empty (non-viable) seed (Neale and Adams 1985). This low level of selfed offspring is consistent with the low levels typically observed in natural stands of conifers; therefore, the presence of multiple ramets of the same clone does not appear to have led to increased levels of ‘selfed’ offspring in this orchard. Because of the high level of pollen contamination observed in this young orchard, it is estimated that genetic gain in the seed crop, relative to what it would be without contamination, is reduced by 43%. In addition, the adaptability of the seed may be questioned. For these reasons, seeds from this orchard should be used with caution at least until the orchard matures further. In the meanwhile, it may well be worthwhile to apply pollen management techniques (e.g., flower stimulation, controlled pollination, supplemental mass pollination) to lessen pollen contamination and increase the genetic quality of seed produced. Such strategies are discussed by Sniezko (1981), Wheeler and Jech (1986), Wiselogel (1986) and El-Kassaby et al. (1989).
Acknowledgements The authors thank the Forest Research Laboratory at Oregon State University (OSU) for providing laboratory and office space and Allan Doerksen for aiding in the laboratory analyses. Yusuf Cengiz (Southwest Anatolia Forest Reseach Institute) helped during the field work in the seed orchard. The study was supported by The Scientific and Technical Research Council of Turkey (TUBITAK) (Project No: TARP-1971) and also by Akdeniz University
415 Scientific Research Projects Unit (Project No: 98.01.0121.06). The TUBITAKBAYG/BDP program granted a seven months research scholarship to Nuray KAYA to study at OSU.
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