MOLECULAR GENE MARKERS FOR RETAINING PLANT BIODIVERSITY

     The end of the second millennium was marked by unprecedented reduction in specific diversity of life. Over the past century, 25,000 higher plant species had disappeared due to human activity. To stop the impending ecological disaster, measures are primarily needed to protect nature from pollution and harmful impacts on habitats of representatives of various species. However, to preserve certain plant species protective measures are no longer enough; today, measures are needed to restore those species. Knowledge accumulated by population genetics indicates that each species has its own inherent, evolutionally formed level of gene diversity. Programs designed to restore species should not ignore historically formed intraspecific genetic subdivision and its variability level. Hence, work designed to restore a given species should include the study of its genetic structure.
     Programs for protecting rare individuals often begin with identifying their taxonomic position. The nature of protective measures would depend on whether the species needing protection is a rare endemic or disappearing population of a widespread species. The morphometry and cytological evidence used therefor long ago are insufficient, since they could depend on the impact of environment on phenotypic display of indications.
      A direct study of representative DNA would be most suitable for elucidating the genetic variability of species and establishing the genetic associations between the representatives examined. At present, the DNA genetic structure is investigated with the aid of various molecular markers. Proteins are used as a measure of genome variability, since they are products of gene expression and can provide information on the structure and state of corresponding DNA sites. Among the various protein markers used in taxonomy and nature-protection measures, allozyme analysis is most widely employed. In recent years, the method based on polymerase DNA chain reaction involving arbitrary primers (RAPD-analysis) has become widespread. It studies DNA loci with the aid of single primers that identify complementary sites on both DNA chains. Unlike the allozyme method, RAPD allows to analyze not only the unique, but also the non-coding DNA portion. Thanks to that, it has been used with success in several laboratories to identify the taxonomy of species and characterize the genetic structure of populations (including that of rare species).
     The Laboratory of Biotechnology, Institute of Biology and Soil Science, F. E. Branch, Russian Academy of Sciences, uses RAPD-analysis to conduct population genetic studies of rare plant species that need protection, as well as widespread species involving complex taxonomic issues, whose resolution would help clarify the evolutional history of species.
     The subjects of study are species, natural populations and cultivated forms of real ginseng, and also continental and insular populations of irises and larchwood populations.
     RussiaТs Maritime Province (Primorye) is the only place on Earth, where real ginseng (Panax ginseng C. A. Meyer) is preserved in natural habitats. An examination of the extent of genetic variability of natural populations should be the theoretical foundation for developing a scientifically grounded program for preserving ginseng. Using several arbitrary primers, we studied the DNA of representatives from three natural ginseng populations originating from Khasan, Spassk and Chuguyevka Districts.  The populations were shown to differ in genetic variability of their representatives, which showed not only in the presence of polymorphous loci in the DNA of several plants, but in varied intensity of homologous fragments in DNA amplification profiles in various plants.  The Khasan population proved virtually monomorphous, while the Chuguyevka and Spassk populations were noted to have changed DNA fragments. Further studies with other plants and primers would help form a definite idea on the genetic structure of still existing natural ginseng populations that would be used in developing protective measures. We also investigate ginseng forms cultivated in nature and in vitro. The obtained results show that cultivation in vitro leads to substantial changes in the DNA of starting forms to render dubious the possibility to preserve DNA versions in cultivated cells.
     Our studies of irises show the possibility of applying RAPD-analysis to resolve complicated issues associated with the status of some taxa. Today, there is no single classification for the Iris genus. Using the RAPD-method, we typified the DNA of representatives of the Russian Far Eastern irises I. setosa, I. ensata, I. laevigata, I. uniflora, I. sanguineae and Pardanthopsis dichotoma. Each of the iris species has its specific spectrum of RAPD-products characterized by a specific set of fragments.  Basing on obtained amplification pictures with primers allowing to distinctly differentiating  the species examined, we plotted their genetic similarity dendrogram. The obtained results allow to state our view on the debatable issue on the status of the species I. uniflora to assign it to the section Limniris. Besides, according to our evidence, the species I. ensata and I. laevigata are genetically remote from each other, and this supports a later view that they possibly belong to different series.
 We also conducted studies and adapted the RAPD-method to population genetic investigations of Larix Mill. We selected a simple and short method for isolating larchwood DNA and effective primers, and optimized the conditions for running poymerase chain reaction.
 

YU. ZHURAVLEV, Corr. Member, Russian Academy of Sciences, Director, Institute of Biology and Soil Science, Far East Branch, Russian Academy of Sciences.

G. REUNOVA, Cand. Sci. (Biology), Institute of Biology and Soil Science, Far East Branch, Russian Academy of Sciences.

M. M. KOZYRENKO, Cand. Sci. (Biology), Institute of Biology and Soil Science, Far East Branch, Russian Academy of Sciences.

E. ARTYUKHOVA, Cand. Sci. (Biology), Institute of Biology and Soil Science, Far East Branch, Russian Academy of Sciences.

T. MUZAROK, Cand. Sci. (Biology), Institute of Biology and Soil Science, Far East Branch, Russian Academy of Sciences.

I.  SAZONOVA,  Junior Researcher, Institute of Biology and Soil Science, Far East Branch, Russian Academy of Sciences.

M. ILYUSHKO, Graduate Student,  Institute of Biology and Soil Science, Far East Branch, Russian Academy of Sciences.

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