June 21, 1999

Identification of Eucalyptus species by DNA analysis of wood chips

Nippon Paper Industries Co., Ltd.

NIPPON PAPER INDUSTRIES CO., LTD. succeeded in identifying Eucalyptus species by the molecular biotechnological analysis of extracted deoxyribonucleic acid (DNA) from wood chips that are materials for the paper manufacturing.
Several species that grow fast in the genus Eucalyptus are commonly used for pulpwood. The genus Eucalyptus includes more than 600 species. As some species morphologically resemble each other, it is difficult to identify the species by observation of the tree form. Each species has different yield of pulping, and only several kinds of Eucalyptus species, represented by Eucalyptus globulus globulus, are fit for pulpwood. Therefore it is a very serious matter which species are chosen for materials from the points of production cost and forest resources conservation in the paper manufacturing industry.
As it was difficult to identify the species of shipped wood chips from oversea to the paper mill in Japan, Nippon Paper Industries had to rely on information from exporters to recognize species of its wood chips. Besides, the ratio, using the waste lumber and useless timber as wood chips to utilize all forest resources effectively, is high in the paper manufacturing industry. Identification method from wood chips has been required for paper manufacturing industry.
DNA is the universal determinants of genetic behavior and consists of the constant region and the polymorphic region between individuals or species. Therefore each species is identified by the analysis of the polymorphic region.
NPI sequenced rbcL gene of several Eucalyptus species.
rbcL gene on chloroplast DNA, code the enzyme for ribulose bisphosphate carboxylase large subunit, is important for carbon dioxide fixation in photosynthesis. In comparison with these DNA sequences, NPI found the polymorphic regions.
Using these results, NPI developed a rapid method for identifying Eucalyptus species by PCR-SSCP* analysis, which is a technique for the detection of minor sequence changes in the DNA fragment.
The method is shown as follows: (1) DNA is extracted from tissue such as wood chips or leaves. (2) The polymorphic region in rbcL gene of extracted DNA is amplified by PCR. (3) PCR products are sifted by SSCP analysis. Polymorphism of PCR products is detected as the differences of mobility of the DNA bands, and each species is visually classified.
Age or conditions of a sample for PCR-SSCP analysis does not affect the data, as a seed, a tree, or a wood chip come from one individual has the same genetic information and DNA is materially stable.
NPI aims to manage the imported wood chips and seeds for plantation to make suite for each products or projects by this method.
NPI has already patented its application in Japan
*PCR-SSCP analysis: Polymerase Chain reaction Single Strand Conformation Polymorphism
PCR The PCR is an in vitro method for the enzymatic synthesis of specific DNA sequence. 20 cycles of PCR yield about a million-fold amplification. One wood chip or a scrap of leaf is enough to prepare DNA for PCR-SSCP analysis.
SSCP The PCR product that is doubled is denatured to form a single strand, and analyzed by a non-denaturing polyacrylamide gel electrophoresis. As DNA has a negative charge, the fragment of DNA migrates to the direction of the positive pole. Single stranded DNA has specific conformation in the gel by intramorecular interaction if there is a sequence change. Single stranded DNA migrates differently by electrophoresis. Polymorphism like this is detected as the difference of mobility of the DNA bands.