GENETIC VERIFICATION OF FOREST REPRODUCTIVE MATERIAL EXAMPLES OF IMPLEMENTATION

Janusz Sabor
Department of Forest Trees Breeding, University of Agriculture in Cracow, Poland

ABSTRACT

The possibility of the genetic verification of forest materials on the basis of morphological and isoenzyme markers and also of genetic markers directly characterizing DNA was evaluated. Against this background the basis of the genetic verification of progeny of forest trees using the Backmann elimination law was presented. Examples were given of using isoenzymes for the identification of populations and genotypes and also of the genetic markers of the RAPD, ALP, PCR-RFLP type and of the micro-satellite DNA for the identification of populations and genotypes of selected species of forest trees.

Key words: Forest Reproductive Material, morphological markers, isoenzymes, terpenes, genetic, DNA markers, mitochondrial and chloroplast DNA, micro-satelites.

INTRODUCTION

Genetic investigation of forest trees currently is the most rapidly developing field of forest sciences. It is based on general laws of population genetics which determine the variability of organisms and on the principles of passing traits on the progeny, i.e. of heredity.

The use of biochemical methods in the genetic identification of the origin of reproductive materials is more and more important owing to the growing trade of seeds and seedlings. The evaluation of the genetic structure of trees using isoenzymes, terpenes and phenols allows for the investigation of the role of mating systems, inbreeding, gene flow between populations, and also of neighbour mating in stands; it also plays a significant role in the taxonomy of trees.

The current possibilities of using modern analytic techniques, chiefly chromatography and electrophoresis, allow for the introduction of reliable tests in the genetic verification of forest reproductive materials.

The results of genetic studies are implemented in the practice of forestry not only in the form of separate projects but also as the basis of normally conducted sylviculture which includes seed and nursery sciences based on genetic principles of population and individual selection of forest trees.

MATERIAL AND METHODS

In the expanding trade of seeds and forest planting stocks it is necessary to use specific methods in verifying the provenance of Forest Reproductive Material. Genetic progeny testing is based on the Backmann exclusion law (1986). This evaluation includes one or more traits whose phenotypic divergence is only based on the genotypic diversity. The application of the exclusion law in the identification of the Forest Reproductive Material is given in Fig. 1.

The genetic verification of single trees and their populations takes into consideration the analysis of marker substances. They chiefly are biochemical compounds produced in the metabolism of forest trees [10]: enzymes and terpene and phenol compounds. Their level and diversity depend on the activity of respective gene loci. The progress in molecular biology also allowed developing genetic markers directly based on differences in the structure of DNA and on this basis populations and single trees could be identified [9]. The verification is based on the varied sequences of nucleotides in the nuclear, mitochondrial and chloroplast DNA.

MORPHOLOGICAL MARKERS

The genetic diversity of a tree population and of single trees can be evaluated by the phenotypic characteristics of differences between numerous traits, e.g., shape and colour of leaves; timing of developmental phases; forms of tree habit etc. The variation is a natural trait of living organisms and concerns single trees; whole populations; or their parts only. In the population genetics the measured or observed value of a trait is the phenotypic value of an individual in which the components of the effect of the genotype, the environment and the G x E interaction  are singled out. The greater is the participation of the genetic effect in the genotypic (total) variation of the investigated  trait, the greater is its diagnostic value in setting apart genetically different partial populations. The value of the genetic markers determines the hereditability coefficient h² sensu lato and sensu stricto.

For example: the spring growth (h² – 0.9); the shape of cones and of cone scales; and the wood density (h²-0.7) in spruce or tree curvature in Scots pine (h²-0.7) can be used as good morphological markers (Table 1, Fig. 2, 3, 4, 5).

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b

BIOCHEMICAL MARKERS

Isoenzymes
With respect of isoenzyme markers the evaluation of the reproduction material at the provenance level is difficult on the account of a great genetic variation in the distribution range of the species and a small variation between the partial populations. Lewandowski [4] claims that apart from a very few populations the evaluation of the genetic provenance divergence is not possible. In Poland the genetic polymorphism of numerous populations has not been evaluated, however these populations frequently constitute the Basic Forest Material of the main forest-forming species, chiefly composed of approved seed stands. On the other hand, the verification of plus trees and clones in seed orchards can be effective. For coniferous species the evaluation of genetic identification is based on the haploidal reserve tissue of the endosperm of seeds. The most polymorphous enzymatic systems are usually selected for analysis: e.g., for Scots pine ADH, GDH, GOT, LAP and MDH; for spruce GDH, GOT and LASP; for fir IDH, LAP and PGM. The evaluation of the genotype of deciduous species is based on analysing the enzymatic systems of diploidic tissue sampled from young dormant buds. This method is limited by the fact that the genome of an individual is evaluated by an indirect investigation of a limited number of genes; a small number of accessible loci.  Its merits are based on the following principles: they are inherited according to the Mendel rules; their character is co-dominant; they represent traits which are irrelevant to the environment; they are ontogenetically stable; and in their identification the simple and relatively inexpensive technique of electrophoresis can be used [9]. Markert and Moller [5] proposed the following interpretation of the isoenzyme variation: each enzyme can be associated with a single gene and every species can possess a unique and characteristic ability of synthesis and arrangement of isoenzymes. Every fringe in a zymogram is a genotypic presentation of a gene confirming the Beadle and Tatum theory of "one gene, one enzyme".

Terpenes
Terpenes are the basic constituents of volatile compounds in essential (resin) oils filling resin ducts of needles, wood and bark of most coniferous species. Of the total number of 60 constituents of the chromatographic spectrum mono hydrocarbons and sesquiterpenes alpha and beta pinene, limonene, myrcen, phellandren and delta 3-caren most frequently are used as genetic markers. In the populations of forest trees the composition of terpenes is variable and their synthesis is controlled by single loci. Currently about 15 such loci are  known. In the area of occurrence of a species the geographical variation of different races is chiefly analysed on the basis of histograms revealing the probability distribution of occurrence of the chromatographically assessed terpene profiles. For example, the characteristic marker for Scots pine is  a "high" or "low" delta 3-caren.  The allele coding the low level of caren in pine is recessive unlike the dominating allele responsible for the "high" caren [16].

Genetic DNA markers
Genetic DNA markers include the genetic code contained in the nuclear, mitochondrial and chloroplast DNA. They should be characterized with allele polymorphism; co-dominance; selection neutrality (lack of link); and independence from environmental conditions. The practical techniques utilize the PCR (Polymerase Chain Reaction) reaction of copying DNA fragments by the Tag polymerase enzyme.

RAPD (Randomly Amplified non Polymorphic DNA)
The RAPD method can be used in the verification of stands (populations) however in this case a high number of starters is needed in evaluating the variation of the genome. In the identification of differences at the allelic level in a locus it is proposed to evaluate haploidic tissues (pre-endosperm in seeds). In general, it is postulated that the less expensive RAPD technique is an introduction to different analyses which more thoroughly investigate the variation of the genome.

AFLP (Amplified Fragment Length Polymorphism)
The AFLP technique allows for analysing genetic distance; identification of cultivars; the purity of seeds; and – on account of the possibility of evaluating numerous polymorphic sequences of the genome with the use of specific starters – it can be used in the identification of the Forest Reproductive Material within the scope of intra-specific variation of forest trees such as Scots pine and pedunculate and sessile oaks. Compared with the RAPD technique, the ALP method can be used in evaluating a higher number of loci and DNA fragments and also sequences of Short Sequence Repeats (SSR) [7].

RFLP (Restriction Fragment Length Polymorphism)
The RFLP technique uses differences in length of DNA fragments whose sequences are recognised by restriction enzymes (e.g., Eco R1). It can be used in the investigation of chloroplast and mitochondrial DNA polymorphism in the taxonomy of trees. The RFLP technique can be used in the identification of the Forest Reproductive Material for related broad-leaved species such as maple, alder, birch, beech, ash and linden. The RFLP methods is applied in the CYTOFOR international EU program of research on the basic forest material and forest reproductive material. In this program analyses of chloroplast DNA covered stands and seeds of 22 deciduous species of Europe [7].

The methods mentioned above do not allow to determine the Forest Reproductive Material from an unknown source, being only applicable to the testing of planting stock and seeds from a known stand which constitutes the Basic Forest Material.

Microsatellites (SSR – Simple Sequence Repeats)
The so-called microsatellites (SSR – Simple Sequence Repeats) are specific DNA markers. They are tandem repeats of sequences 1–6 nucleotides in length. Their merits are high polymorphism; usability of all tissues; high repeatability; and genetic determinism. Because of the methods of hereditability microsatellites are classed as nuclear and cytoplasmic (chloroplast and mitochondrial).

In coniferous trees chloroplast DNA (cpDNA) is inherited paternally and the mitochondrial DNA (mtDNA) – maternally. In broad-leaves species cpDNA and mtDNA are maternally inherited.

RESULTS AND IMPLEMENTATIONS

Genetic basis of establishing seed plantations in Poland
Since 1963 seed plantations established on the principles of population and individual genetics of forest trees are included in the project currently carried out in state forests as the program of "Selection and preservation of gene resources and selection breeding of forest trees in Poland" (Fig. 5 a, b.). The utilization of seeds from seed plantations are estimated at 10–13%.

In seed plantations the basic genetic assumption is the panmixis mating of individuals, however, it is rarely confirmed in practice owing to the occurrence of self-pollination, pollution with alien pollen, divergent blooming phenology and also to the wrong distribution of grafts. The valuation of the genetic content of a plantation also depends on the degree of its isolation and size. It is accepted that in a plantation the stand is represented at least by 40 clones of pine and at least by 30 clones of the remaining species. In Poland all the plantations are included in the national test program. In testing stands each seed plantation must be represented by the progeny of 50% clones at least [11]. In evaluating the genetic polymorphism isoenzymic markers and also markers of the RAPD, AFLP, PCR-RFLP type and microsatellite DNA are most frequently used [2,4].

The above given methods allow for determining the degree of pollution with alien pollen in the seed plantation and the level of self-pollution of the clones; they also identify the genetic similarity of grafts and seeds harvested in the population.

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b

Estimation of genetic value of Norway spruce stands of the Silesian Beskid range
The investigations conducted in the years 2003-2005 on the genetic value of stands of the Silesian Beskid range and also of stands in different mountain forest districts included in the program of conversion (the Zywiec Beskid range) covered:

• the evaluation of adaptability traits of spruce and other forest-forming species in the Beskid ranges (fir, beech) and admixture species on experimental plots, on the basis of analysis of the genetic-breeding value of progeny and also the evaluation of the genetic structure of selected Beskid stands determined on the basis of isoenzyme and terpene markers;

• development of rational principles and stages of the conversion of spruce stands in the Silesian and Zywiec Beskid ranges on the basis of their genetic value;

• evaluation of the stage of collecting and the feasibility of using the stocks and deposits of seeds, which meet the genetic principles of conversion of spruce stands in the Silesian and Zywiec Beskid ranges (the Forest Reproduction Material) on the basis of stations of the Regional Gene Bank in the Wisla forest district.

The conversion of the Silesian Beskid and also Zywiec Beskid forest stands is based on the assumption that in the composition of stands of these regions spruce participation should be greatly reduced while the populations of indigenous origin and spruce stands whose high genetic quality was confirmed in progeny tests should be preserved. The Istebna spruce is such a population.

The genetic-breeding value of Beskid stands was evaluated in provenance tests; the optimum seed base for fir and beech and for admixture species was determined. The evaluation also concerned the genetic structure of selected approved seed stands of Istebna spruce using the method of isoenzyme markers [8] and the method of genetic terpene markers [13].

The presented analyses of the genetic structure of Beskid spruce confirm previous estimates concerning genetic heterogeneous quality of the partial spruce populations of the Istebna race, this being an indispensable condition of the  proper selection of the Forest Basic Material and Forest Reproduction Material for the conversion of stands and selective collection of stocks and deposits in the Carpathian Gene Bank Station in the Wisla Forest District.

The investigation carried out by Polak-Berecka [8] allowed for the evaluation of the genetic structure of Istebna spruce, i.e. 11 loci: GOT, PGI, GHD, MNR, PGM, IDH, ACO, MHD, SKDH,  LAP, and 6-PGDH for 37 plus trees of Istebna spruce.

The investigated loci were homozygous in most cases and their alleles were typical for the species. MNR-B, PGI-A and MDH-B loci were monomorphous while in the remaining polymorphous gene loci two to six alleles were identified. In ACO-A and MDH-A loci the occurrence of new alleles not described in spruce populations of Central Europe yet, was recorded. The genetic interpretation of zymograms showed that in the investigated population of Istebna spruce from Zapowiedz and Malinka Forest Ranges the level of heterozygosis was low; isoenzymic loci were mostly homozygous while the distribution of genetic variants was not uniform.

At the same time the evaluation of the genetic structure of Carpathian fir  carried out in the wide scope of 29 gene loci [6] allowed for the progeny verification of the Forest Reproductive Material of this species. Studies are also advanced on the use of terpene markers in evaluating genetic polymorphism and in this number on the plastic provenance of fir from Powrożnik [13].

CONCLUSIONS

1. The most useful method of identifying the Forest Reproductive Material using analysis of DNA polymorphism is the PCR-RFLP technique owing to the high level of detecting genetic similarity of the investigated individuals; the fairly low cost of analyses; and  the high degree of reliability [7].

2. At the current level of research the development of micro-satellite methods allows for the identification of plus trees and their vegetative (strains, clones) and generative (seeds) progeny. In general opinion [2] they are the best genetic markers of the Forest Reproduction Material.

3. It is recommended to develop much less expensive morphological and physiological markers for the identification of the Forest Reproduction Material.

REFERENCES

1. Backmann F., 1986. Genetic means of Verifying Observance of the Law. I. Methodical principles of "provenance identification" IUFRO Joint Meeting of WP.S.32.03–14 on Biochemical Genetics and Legislation of Forest Reproductive Material. Bundesforschung for Forst und Holzw. Hamburg.

2. Burczyk J., 2003. Wykorzystanie markerów mikrosatelitarnego DNA do identyfikacji populacji i genotypów [The use of microsatellite DNA markers In the identification of populations and genotypes] W: Opracowanie szczegółowych wymagań wynikających z Dyrektywy Rady 1999/105/WE z dnia 22 grudnia 1999 r. w odniesieniu do leśnego materiału rozmnożeniowego. IBL Warszawa [in Polish].

3. Giertych M., 1989. Doskonalenie składu genetycznego populacji drzew leśnych [Improvement of the genetic composition of forest tree populations]. Wydawnictwo SGGW-AR, Warszawa [in Polish].

4. Lewandowski A., 2003. Wykorzystanie izoenzymów w identyfikacji populacji i genotypów [Use of isoenzymes in the identification of populations and genotypem] W: Opracowanie szczegółowych wymagań wynikających z Dyrektywy Rady 1999/105/WE z dnia 22 grudnia 1999 r. w odniesieniu do leśnego materiału rozmnożeniowego. IBL Warszawa [in Polish].

5. Markert C.L., Moller F., 1959. Multiple forms of enzymes: tissue, ontogenetic and species specific patterns. Proc. Nat. 1.Acad. Sci. (USA), 45, 753–763.

6. Mejnarowicz L., 2002. Genetic analysis of fir populations in the Beskid Mts. IUFRO Conference Trippstadt. Germany.

7. Nowakowska J., 2003. Wykorzystanie markerów genetycznych typu RAPD, ALP, PCR-RFLP do identyfikacji populacji i genotypów wybranych gatunków drzew leśnych [The use of genetic markers of the RAPD, ALP and PCR-RFLP type in the identification of populations and genotypes of selected species of forest Teres] W: Opracowanie szczegółowych wymagań wynikających z Dyrektywy Rady 1999/105/WE z dnia 22 grudnia 1999 r. w odniesieniu do leśnego materiału rozmnożeniowego. IBL Warszawa [in Polish].

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11. Sabor J., 2004 (Editor). Program testowania potomstwa wyłączonych drzewostanów nasiennych, drzew doborowych, plantacji nasiennych i plantacyjnych upraw nasiennych [Program of progeny testing of approved seed stands, plus trees, seed plantations and field seed growing]. Dyrekcja Generalna Lasów Państwowych. Warszawa [in Polish].

12. Schmidt-Vogt H., 1977: Die Fichte, Bd 1, Ed. Paul Parej Verlag, Hamburg, Berlin.

13. Skrzyszewska K., 2004. Ocena struktury genetycznej wybranych populacji swierka pospolitego Beskidu Śląskiego metodą markerów terpenowych [Evaluation of the genetic structure of selected spruce populations in the Silesian Beskid Mts., using the method of terpene markers].W: Doskonalenie rewitalizacji siedlisk i przebudowy drzewostanów górskich w RDLP Katowice z uwzględnieniem poprawy stosunków wodnych i selekcji genetycznej drzew. Etap II. Maszynopis. Kraków [in Polish].

14. Staszkiewicz J., 1993. Zmienność morfologiczna szpilek, szyszek i nasion [Morphological diversity of needles, cones and seeds]. W: Biologia sosny zwyczajnej. PWN Instytut Dendrologii PAN. Sorus Poznań-Kórnik, 33–43 [in Polish].

15. Swoboda P., 1936. O systematicke hodnote kury nagich drevin, zejmena o kure smrku a jej mutacjach [Systematic (hodnote?) of bare trees, zejmena of spruce bark and its mutations]. Zvlastni otsik c casopisu Legnicka prace, vydavaneho Cs mativi lesnickou v Pisku [in Czech].

16. Tigerstedt et al., 1978. Inheritance and genetic variation of monoterpenes In Scots pine (Pinus sylvestris L.). In: Prof. Conf.on Biochemical Genetics of Forest Trees. Umea, 293–299

Accepted for print: 14.12.2009

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