Kemerovo, Kemerovo, Russian Federation
A general description of molecular genetic markers is provided in the present article. The classification of DNA markers used for identification of raw materials of plant origin is presented. The most appropriate method for identifying raw materials derived from fruit and berries is chosen using study reports. The use of PCR for determining the quantitative and qualitative composition of the raw materials is considered. DNA regions used for PCR diagnostics of raw material derived from fruit and berries are characterized. The significance of amplification for a PCR test is outlined. The optimal PCR conditions have been selected and the advantages of this method have been revealed in the present study. Amplification profiles of DNA from the samples have been analyzed using different primers. Experiments with different primers allowing for identification of the raw material were carried out. The possibility of using a complex including a common gene and a variable gene for the identification of raw materials derived from fruit and berries has been considered. The sequence of DNA regions has been analyzed. The possibility of using a ribosomal RNA gene for generic and interspecific differentiation of DNA samples has been demonstrated. The significance of oligonucleotide primers and PCR product length for the reliability of the whole genotyping system has been elucidated. A scheme of PCR-based DNA profiling has been developed. Two types of the procedure were compared and the most appropriate type was chosen according to cost efficiency.
DNA, genome, identification, genetics, primers, amplification, PCR amplicons, amplification profile
INTRODUCTION
Identification of plant species from which raw material was derived is among the main areas of application of molecular genetic markers. DNA fragments associated with a specific nucleotide sequence constitute a new class of molecular markers. The number of such markers is severalfold higher than that of the markers characterized previously (isozymes, storage proteins, and morphological features). Besides, expression of DNA markers is neither dependent on the phenotype nor tissue-specific and can be detected at any stage of plant development. The use of DNA markers led to changes in the methods of evaluation of genetic diversity of plants, certification and classifica-tion of plant varieties, and genetic monitoring and selection breeding [4].
All studies related to identification of plant samples are based on the assumption that DNA fragments with equal molecular weights and the same activity represent the same genomic fragments within one family of plants. Specific resolved DNA bands are used as tools for the assessment of the level of similarity between multiband DNA profiles. The possibility of detecting identical multiband DNA profiles for two randomly selected individuals is close to 2•10-9 for avocado, 1.5•10-9 for papaya varieties, 4.2•10-5 for apple varieties, and 2.4•10-3 for varieties of raspberries and blackberries [12, 13].
Identification of plants is carried out stepwise in a certain direction:
1. identification of individuals;
2. detection of hybrid forms;
3. investigation of pedigrees of plant specimens.
The purpose of identification of individuals is assignment of a plant specimen to a species, subspecies, variety, etc. or finding a solution for a taxonomic problem.
The main areas of use of molecular genetic markers are the following:
- identification of species, varieties, and other forms of plants;
- assessment of genealogical relationships between plants;
- search for molecular genetic markers associated with desirable traits.
DNA markers must meet several requirements, such as:
- availability of phenotypic manifestations of allelic variants for the identification of different individuals;
- difference between allele replacement at one locus and those at other loci;
- availability of a substantial part of allelic substitutions in the target locus for identification;
- random character of the sample of genetic loci investigated with regard to physiological effects and the degree of variability;
- uniform distribution in the genome;
- relative neutrality.
There are no primers that would meet all these requirements [4, 9].
Molecular genetic markers that are most frequently used in practice can be divided into the following classes:
- markers expressed as visible morphological characteristics;
- markers constituted by structural portions of genes encoding the amino acid sequences of proteins;
- markers constituted by non-coding regions of structural genes;
- markers constituted by various DNA sequences, for which the relation to the structural genes is usually unknown – in other words, short repeats spread throughout the genome (RAPD – randomly amplifiable polymorphic DNA; ISSR – inverted short sequence repeats; and RFLP – restriction fragment length polymorphism), microsatellite loci (tandem repeats of a unit consisting of 2–6 nucleotides), and others.
1. Altukhov, Yu.P., Geneticheskie protsessy v populyatsiyakh: ucheb. posobiye (Genetic processes in populations: a study guide) (Akademkniga, Moscow, 2003).
2. Biryukova, V.A., Zaitsev, V.S., Pankin, A.A., et al., DNK-genotipirovanie kartofelya i ego dikorastushchikh sorodichei na osnove polimorfizma umerennykh povtornostei semeistva R173 (DNA genotyping of potato and its wild relatives based on polymorphism of moderate repeats of the family R173), in Materialy Mezhdunarodnoy yubileynoy nauchno-prakticheskoy konferentsii: Nauchnyye trudy. (Materials of International jubilee scientific and practical conference: Proceedings), Minsk, 2003. Part 1. P. 313.
3. Kil’, V.I., and Gronin, V.V., Geneticheskie markery chuvstvitel´nosti populyatsiy koloradskogo zhuka k transgennomu kartofelyu (Genetic markers of sensitivity of the Colorado potato beetle populations to transgenic potatoes), Nauka Kubani (Kuban’ Science), 2005. № 4. P.126.
4. Komarova, I.N., Razrabotka PTsR-test-sistem dlya vidovoi identifikatsii i kolichestvennoi otsenki myasnogo syr´ya v sostave melkoizmel´chennykh polufabrikatov i gotovykh myasnykh produktov (Development of PCR test systems for species identification and quantification of raw meat in of semi-finished and finely ground meat products, Dis…kand.tekhn.nauk (Diss.… Cand. Sci. (Tech.), Moscow, 2005.
5. Lewin, B., Geny (Genes); translated into Russian by Gintsburg, A.L. and Ilyin, T.S. (Mir, Moscow, 1987).
6. Palilova, A.N., Urbanowich, O.Yu., Dolmatovich, T.V., et al., Poisk molekulyarnykh markerov ustoychivosti rasteniy kartofelya k virusnoy infektsii (Search for molecular markers of potato plant resistance to virus infection), in Materialy Mezhdunarodnoy yubileynoy nauchno-prakticheskoy konferentsii: Nauchnyye trudy. (Materials of International jubilee scientific and practical conference: Proceedings), Minsk, 2003. Part 1. P. 316.
7. Politov, D.V., Primenenie molekulyarnykh markerov v lesnom khozyaistve dlya identifikatsii, inventarizatsii i otsenki geneticheskogo raznoobraziya lesnykh resursov (Application of molecular markers in forestry for identification, inventory and assessment of genetic diversity of forest resources), Lesokhozyaystvennaya informatsiya (Forestry Information), 2008. V. 34. P. 24.
8. Prosekov, A.Yu. and Babich, O.O., Gennaya inzheneriya: uchebnoe posobie (Genetic engineering: a tutorial) (Redaktsiya zhurnala “Dostizheniya nauki i tekhniki APK”, Moscow, 2010).
9. Romanova, O. V., Identifikatsiya sortov kostochkovykh kul´tur s pomoshch´yu PTsR-analiza. (Identification of the varieties of stone fruit plants by PCR analysis), Dis.... kand. sel´.-khoz. nauk (Diss. Cand. Sci (Agriculture)), Moscow, 2007.
10. Coyne, V.E., James, M.D., and Reid, Sh.J., Molecular biology techniques manual: standard PCR protocol. 1994.
11. Frey, J.E., Genetic flexibility of plant chloroplasts, Nature, 1999. V. 398. P.115.
12. Fulcrand, N., Cheynier, V., and Oszmianski. J., An oxidized tartaric acid residue as a new bridge potentially competing with acetaldehyde in flavan- 3 -ol condensation, Phytochemistry, 1997. № 46. P. 223.
13. James, S.A., Collins, M.D., and Roberts, I.N., Use of an rRNA internal transcribed spacer region to distinguish phylogenetically closely related species of the genera Zygosaccharomyces and Torulaspora, Int. J. Bacteriol., 1996. № 46. P. 189.
