Báo cáo khoa học: "The mating system in a Scots pine clonal seed orchard in Poland"

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Original article mating system The in a Scots pine clonal seed orchard in Poland J Burczyk Polish Academy of Sciences, Institute of Dendrology, Department of Genetics, Kornik, Poland 62 - 035 13 November (Received 1990; accepted 8 April 1991) Summary — The mating system of a clonal seed orchard of Pinus sylvestris L consisting of 32 clones was studied using allozyme marker loci. Single-locus estimates of outcrossing rate ranged from 0.907-1.106 (with a mean of 1.011), while the multilocus estimate was 0.987. The outcrossing rates of individual trees ranged from 0.840-1.208. The variance effective population size estimated for males was equivalent to 14 individuals. Some patterns of non-random mating were found. The level of inbreeding as measured by average fixation indices was low, both in the parental and the fil- ial generations. The usefulness of Scots pine seed orchards in forest tree improvement has been discussed in the light of genetic efficiency. Pinus L / seed orchard / / variance effective sylvestris / outcrossing inbreeding population size Résumé — Système de croisements dans une pinède de clones de pins sylvestres, issus de Pologne. Le système de croisements dans une pinède de Pinus sylvestris composée graines, en de 32 clones est étudié grâce à des locus d’allozymes marqueurs. On estime, d’après le taux de croisements, les locus uniques à 0,907-1,106 (moyenne :1,011) et les locus multiples à 0,987. Les taux de croisements d’arbres considérés individuellement sont de 0,840-1,208. On évalue la popula- tion de mâles utilisée pour la détermination de la variance à 14 individus. On trouve quelques mo- dèles de croisement non aléatoire. Le niveau d’inbreeding, mesuré par les index de fixation moyenne, est faible, aussi bien dans les générations parentales que dans les générations filles. L’utilité pour l’amélioration des arbres forestiers de pinèdes de pins sylvestres dont les individus sont issus de graines est discutée en termes d’efficacité génétique. Pinus sylvestris L / pinède à individus issus de graines / croisement / inbreeding / taille de la population servant au calcul de la variance
INTRODUCTION El-Kassaby et al, 1989; Muona and Harju, 1989). Information on seed orchard mating systems analyzed based on multilocus Seed orchards are established to promote methods for other conifers is limited the production of seeds of high genetic (Adams and Birkes, 1991).Mating systems value. The clones used in such plantations of individual trees have also been studied are phenotypically or genetically selected using multilocus methods (Shaw and Al- from natural or artificial stands, and are lard, 1982a; Neale and Adams, 1985; Rit- replicated many times as grafts. It is as- land and El-Kassaby, 1985; Koski and Mu- sumed that full panmixis occurs within an ona, 1986; Perry and Dancik, 1986; El- orchard and that all clones make an equal Kassaby et al, 1987; Moran et al, 1989). contribution to the filial generation. Howev- The assumption of the estimation of popu- er, many studies have demonstrated non- lation outcrossing rate is that the outcross- random mating in seed orchards of some ing rate is constant among individuals in conifers (Cheliak, 1984; Müller-Starck, the population. However, heterogeneity of 1984; Barrett et al, 1987) and thus the outcrossing among trees has been ob- evaluation of genetic efficiency of seed or- served in seed orchards (Shaw and Allard, chards is of great practical importance in 1982a) and natural populations (Neale and forest tree improvement. Many seed or- Adams, 1985). Apart from the theoretical chards established in Poland in the early value of such studies they are also of prac- 1970s have reached the commercial cone tical interest, especially when open- crop production stage. Breeders are trying pollinated seeds collected from individual to design the next generation of seed or- trees are used for reforestration. chards and more information is required to Many conifers, including Scots pine, ex- understand the processes of mating occur- hibitstrong inbreeding depression (Koski, ring in such artificial populations. To de- 1973) and estimates of the mating system scribe seed orchard efficiency, it is useful parameters are important for detecting to study the levels of inbreeding and pro- non-random mating events that increase portion of individuals effectively contribut- the levels of inbreeding. Knowledge about ing to the filial generation. the rates of selfing would allow breeders to Scots pine (Pinus sylvestris L) is the predict losses caused by inbreeding de- species with which most seed orchards pression, following self-fertilization, when have been established in Europe. The in- seeds from a particular seed orchard are tensity of male and especially female flow- used for reforestration. ering, and the relative stability of high cone crops from year to year result in Scots pine seed orchards still being regarded by MATERIALS AND METHODS foresters as valuable and useful sources of seeds. The mating system of Scots pine seed orchards has been studied in recent The Gniewkowo clonal seed orchard was estab- lished by the Torun Regional State Forest Ad- years (Rudin and Lindgren, 1977; Shen et ministration in 1972 and is located 30 km = al, 1981; Müller-Starck, 1982; Rudin and south-west of Torun, Poland. The site was an Ekberg, 1982; Rudin et al, 1986), and the agricultural field and the nearest Scots pine estimates of outcrossing levels ranged stands are located 1.5 km away towards the from 0.84-0.98. When efficient multilocus north-west. Scions were collected from 32 se- methods were used, the estimates ranged lected Scots pine, plus trees growing in 3 forest districts in the Tuchola forests. Grafts were out- from 0.91-0.977 (Koski and Muona, 1986;
sumption of a mixed mating model, where the planted at 5 x 5 m spacing. The orchard was ar- ranged in 3 blocks. Each block was 0.935 ha in probability of an outcross is independent of size and contained all the clones randomly dis- genotype of the maternal plant (Shaw et al, tributed in different proportions (see Burczyk, 1981).Allele frequencies in the outcross pollen 1990 for details). pool were estimated based on 20 seeds. In January 1989 cones were collected from Spatial homogeneity of allele frequencies in several branches of a single ramet of each of the pollen pool was studied across homozygous the 32 clones from 1 block. Extracted seeds trees by way of a χ analysis (Brown et al, 2 (embryo and megagametophyte tissues) were 1975). Homogeneity of maternal and paternal analyzed using standard electrophoretic tech- genetic contribution into the gene pool was eval- niques (Yeh and O’Malley, 1980). Three enzyme uated by means of a χ test, the frequency 2 systems were assayed by starch gel electropho- counts obtained from the progeny being the ob- resis: aspartate amino-transferase (EC 2.6.1.1; served values of ovule and pollen alleles (Bar- AAT), glutamate dehydrogenase (EC 1.4.1.2; rett et al, 1987). GDH), malate dehydrogenase (EC 1.1.1.37; The variance effective population size was MDH), and 6 variable gene loci were recorded in estimated based on allele frequencies estimated both seed tissues (Aat-1, Aat-2, Aat-3, Gdh, in the mature and filial generations. The method Mdh-3, Mdh-4). For the MDH enzyme loci the proposed by Yasuda was used (Yasuda, 1969; notation presented by Müller-Starck (1984) was after Cheliak et al, 1985). The actual breeding used. The extraction and staining buffers were size is reduced to a value equivalent to the num- according to Yeh and O’Malley (1980) with slight ber of individuals in the idealized theoretical modifications. population, exhibiting the same increase in ho- mozygosity as in the observed population (Crow and Kimura, 1970). In conifers it is possible to Data analysis estimate separately the allele frequencies in ma- ternal and paternal gametes contributed to the next generation; thus the effective number of fe- seeds were analyzed electrophoretically Twenty males and males can be obtained. However, in for each of the 32 clones. An additional 40 the studied case the effective number of fe- seeds were analyzed for each of 16 clones cho- males could not be estimated, because equal sen based on the probability of detecting out- number of seeds per female was analyzed, and cross pollen gametes (see below); thus the mat- only male gametes could be considered as a ing system of individual trees was studied based random sample. on 60 seeds. The numbers of seeds for popula- The frequencies of genotypes observed in tion and individual tree mating system parame- mature and the filial generation were compared ters were constant per clone to arrive at a prop- to that expected under panmixis (Hardy- er mean estimate. The single-locus (t and ) s Weinberg equilibrium) based on a G-test (Sokal multilocus (t levels of outcrossing for the pop- ) m and Rohlf, 1973). The extent of inbreeding was ulation, and the multilocus outcrossing rate for estimated by means of Wright’s fixation index, individual trees (t were estimated based on a ) mi using the formula: F =1-H where H is ob- , e /H o o mixed mating model using the maximum likeli- served heterozygosity and H is expected hetero- e hood procedures and programs described by zygosity according to Nei (1975). The variances Neale and Adams (1985). The clones for estima- of the F indices were obtained by the method of tion of individual tree outcrossing rates were Brown (1970). chosen based on the conditional probability of detecting outcross pollen gametes calculated for When self-fertilization is the sole factor affect- each parental genotype after single-locus esti- level of inbreeding an expected equilibrium ing mation (Neale and Adams, 1985). When this coefficient can be calculated: F e (1-t + )/(1 m = probability is relatively small a great number of t where t is the multilocus outcrossing rate ), m m seeds needs to be analyzed. The planned size (Allard et al, 1968). A substantial difference be- of an analyzed progeny for outcrossing estima- tween F and F indicates that inbreeding other e tion of individual trees was not very large, and than through selfing occurs in the population thus only those 16 clones were chosen for (Shaw and Allard, 1982b). All tests of heteroge- which the conditional probabilities were the high- neity were made using &2 according to Rao chi; est (> 0.7). This could be made based on an as- (1973).
RESULTS pool were spatially homogeneous across sampled trees for all polymorphic loci. the Excess of heterozygosity was observed estimates of outcrossing Single-locus (t ) s in the parental population and the average rate ranged from 0.907 for the locus Mdh-3 Wright’s fixation index was calculated to be to 1.106 for the Gdh locus, with an aver- -0.199 (table III). This excess was statisti- age of 1.011, and significantly were not cally significant for Aat-2 and Gdh. In the studied loci (ta- heterogeneous over the filial population significant excess of hetero- ble I). The multilocus (t estimate of out- ) m detected for Gdh and Mdh-4, zygotes was crossing was 0.987 and was lower than but significant deficiency of heterozy- a the average of single locus estimates. Mul- gotes was observed for the Aat-3 locus (ta- tilocus estimates of outcrossing rate for in- ble III). dividual trees (t ranged from 0.840 for ) mi clone No 214 to 1.208 for clone No 232 and were not significantly heterogeneous DISCUSSION across the studied clones (table II). The average was calculated to be 1.017 and was higher than the multilocus outcrossing The investigations indicated a high propor- rate estimated for the population. tion of outcrossing in the seed orchard ex- Analysis of the variance effective popu- amined. Previous studies in Scots pine seed orchards showed that the outcrossing lation size indicated that the actual breed- ing size of males is equivalent to 14 indi- rate varied widely (0.84-0.988; see table IV). However, most of them were made on viduals contributing gametes to the next generation. When the maternal genetic individual clones using the unique marker- contribution into the gene pool was com- alleles method: thus they cannot be con- sidered population estimates. Results on pared with the paternal one significant dif- ferences were observed for loci Aat-2 the mating system calculated by the more 2 (χ 6.00, P < 0.05) and Gdh (2 14.04, χ efficient single-locus or multilocus methods = = have only been reported by Koski and Mu- P< 0.001). Allele frequencies in the pollen
(1986) El-Kassaby et al (1989) and ona Muona and Harju (1989). The presented multilocus estimate of outcrossing (t 0.987) is very close to m = the multilocus estimates reported for 2 Finnish seed orchards of Scots pine (Muo- na and Harju, 1989). But the average of single-locus estimate is greater (t 1.011) s= than other previously reported analogous estimates (table IV). Comparison of single-locus and multilo- cus population estimates allows inference about the amount of inbreeding other than through selfing in populations (Shaw and Allard, 1982a). The mixed mating model assumes that if matings occur among ge- netically related trees, single-locus esti- mates are biased downward. This has of- ten been observed for natural populations (Neale and Adams, 1985; El-Kassaby et al, 1987; Muona and Harju, 1989) and has explained mainly by the family struc- been ture of a stand. In thisstudy the mean of single-locus estimates is greater than the
the unique marker alleles method is used, multilocus Seed orchards artificial one. are populations created by foresters and be- t-values never exceed unity and only the cause of permutated neighborhood distri- lower-bound estimates of t can be com- mi bution of grafted clones within a plantation pared. The lowest reported values of out- they are expected to be free of the family crossing obtained by the unique marker al- leles method and presented in this study structure or isolated mating groups, except are very similar. Individual tree outcrossing for plantations arranged according to a rates obtained in this study fell within the systematic layout (Giertych, 1971).Thus the single-locus estimates of a seed or- range of variation of the individual out- chard reflect selfing only, without any other crossing rates based on the multilocus form of inbreeding (Barrett et al, 1987). method reported by Koski and Muona (1986) (table IV), confirming low variation Individual tree multilocus estimates (t ) mi of outcrossing among clones. could be compared to values obtained by the unique marker alleles method, be- It is interesting that the averages of indi- cause both methods evaluate the out- vidual tree outcrossing rates are very often crossing level for individual clones. When greater than the multilocus estimates for a
gains (Perry and Dancik, 1986). The ge- population (Neale and Adams, 1985; Rit- netic reasons of self-fertility variation are land and El-Kassaby, 1985; El-Kassaby et quite complicated and cannot be fully ex- al, 1987; and the present study), which is usually due to the influence of some trees plained. with an outcrossing rate estimate exceed- Variance effective population size esti- ing unity. For multilocus estimates values mated for males showed that it is equiva- of outcrossing greater than one are possi- lent to 14 individuals in an ideal theoretical ble if the proportion of detectable out- population, all with an equal opportunity to crosses in the progeny sample is greater mate. Fourteen effective males represent than that expected on the basis of detec- 44% of the actual number of males. The tion probability for an individual tree (Neale imbreeding effective population size for 2 and Adams, 1985). This biologically unrea- Finnish orchards of Scots pine was esti- listic value observed for different loci (table mated to be 66% and 93% of the actual I) or individual trees (table II), could be due number of clones (Muona and Harju, to negative assortative mating or could just 1989). The effective population size of zy- be due to sampling error (Brown et al, gotes derived from seed orchards is prob- 1984), especially when t is near 1 and ably most affected by asynchronous phe- sample sizes are small (Adams and nology and asymmetrical male and female Birkes, 1991).The negative assortative Müller-Starck, (Cheliak, 1984; fertility mating occurs when individuals of particu- For a Douglas fir seed orchard it 1984). mate with each other less lar was shown that estimates of outcrossing genotypes than expected under panmixis, frequently for a group of clones with an intermediate and it could be an effect of phenological reproductive phenology were the highest, differences in male and female flowering indicating maximum panmixis during the within and among trees (Sarvas, 1962). height of flowering (El-Kassaby et al, This could probably be more clearly mani- 1988). However, flowering was not investi- fested for single trees than for a whole gated in this study. population. Each individual tree in a stand The level of inbreeding as measured by is surrounded by the trees of different average fixation indices was low in the pa- genotypes. When differences in flowering rental and filial generations. Lack of in- among them exist, which is common for breeding in the progeny is in line with the most conifers including Scots pine (Jons- negative assortative mating events detect- son et al, 1976), pollen from only a few ed in some cases. The observed genotypic clones is effective in the fertilization pro- distribution, compared to that expected un- cess, influencing the system of mating of der Hard-Weinberg equilibrium showed a individual trees. The average of individual significant excess of heterozygotes in both tree estimates are in fact female estimates generations for some loci. Since the clones of outcrossing rate (Ritland and El- are phenotypically selected genotypes, de- Kassaby, 1985) and could often differ from parture from Hardy-Weinberg equilibrium is the population outcrossing average, which not unexpected for the mature trees. When is identical for males and females (Ross the whole seed orchard was considered as and Gregorius, 1983). Variation among a population consisting of 1 056 grafts, the trees in self-ferility may complicate analy- departure was very significant for all loci sis of data from wind pollinated progeny except Aat-1 (Burczyk, 1990; in press). tests. Progeny of trees with low outcross- However, random mating would bring loci back into equilibrium in the filial genera- ing rates may cause overestimation of ad- tion. The excess of heterozygotes in the ditive genetic variance and potential genetic
WT Adams for the computer programs used in progeny and the differences between ma- mating system estimation and A Lewandowski ternal and paternal contribution into the for his help in developing the electrophoretic gene pool are evidence of some type of techniques. The technical assistance of A Pieku- non-random mating patterns or of selec- ta and H Przybyl is greatly appreciated. tive mortality of homozygous embryos. Similarity between F and F and lack of e mating among relatives suggest that self- REFERENCES fertilization is the main factor affecting in- breeding in the studied population. Equili- Adams WT, Birkes DS (1991) Estimating mating brium populations exhibit a relationship be- patterns in forest tree populations. In: Proc tween Wright’s fixation index (F) and out- Int Workshop on Plant Biology. Biochemical Markers in the Population Genetics of Forest crossing rate (t) according to formula F = Trees. Inst Agrofor, Nat Res Council Italy (1 - t) / (1 + t) (Allard et al, 1968), thus t = (CNR), Porano-Orvieto, Italy, Oct 11-13, (1 F) / (1 + F). Observed fixation index of - 1988 (in press) -0.014 (table III) in the progeny suggests Allard RW, Jain SK, Workman PL (1968) The an outcrossing rate of 1 028, which is very genetics of inbreeding populations. Adv Gen- close to the mean of single-locus esti- et 14, 55-131 s 1.011) (table I). Thus single- mates (t = Barrett JW, Knowles P, Cheliak WM (1987) The locus outcrossing estimates of seed or- mating system in a black spruce clonal seed chards reflect selfing only without any oth- orchard. Can J For Res 17, 379-382 er form of inbreeding (Barrett et al, 1987). Brown AHD (1970) The estimation of Wright’s The high out-crossing rate, homogene- fixation index from genotypic frequencies. ity of the pollen pool, and relatively great Genetica 41, 399-406 number of males and females effectively Brown AHD, Matheson AC, Eldridge KG (1975) contributing gametes to the next genera- Estimation of the mating system of Eucalyp- tion are positive characteristics of the ana- tus obliqua L’Herit by using allozyme poly- morphisms. Aust J Bot 23, 931-949 lyzed population, but the observed pat- terns of non-random mating are not. Brown AHD, Barrett SCH, Moran GF (1985) Mating system estimation in forest trees: Background pollination from pollen parents models, methods and meanings. In: Popula- outside of the orchard was not investigat- tion Genetics in Forestry (Gregorius HR, ed) ed in this orchard and it may increase the Springer-Verlag, Berlin, 32-49 rate of outcrossing of a seed orchard. The Burczyk J (1990) Struktura genetyczna plantacji presented results and some other studies nasiennej sosny zwyczajnej (Pinus sylvestris (Rudin et al, 1986; Muona and Harju, L). Arbor Kórnickie 35 (in press) 1989) allow the conclusion that Scots pine Cheliak WM (1984) Mating system dynamics in seed orchards can be regarded as a a Scots pine seed orchard. In: Proc IUFRO source of genetically valuable seeds; thus Working Party on Ecological Population Ge- establishment of further generations of netics. Göttingen, 1984, 107-117 seed orchards is needed, and seems Cheliak WM, Pitel JA, Murray G (1985) Popula- promising for this species. tion structure and mating system of white spruce. Can J For Res 15, 301-308 Crow JF, Kimura M (1970) An Introduction to ACKNOWLEDGMENTS Population Genetics Theory. Harper and Row, NY This research was supported by Grants from El-Kassaby YA, Meagher MD, Parkinson J, Port- the Polish Forest Research Institute and the lock FT (1987) Allozyme inheritance, hetero- Polish Academy of Sciences. The author tanks zygosity and outcrossing rate among Pinus
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