Page 125 - Zbornik_Konf_2019

https://doi.org/10.15414/2019.9788055220703

4 International Scientific Conference Abstracts Book
th
INTRON LENGTH POLYMORPHISM OF ß‐TUBULIN AND ACTIN GENES AS EFFICIENT
TOOL FOR CAMELINA SATIVA (L.) CRANTZ. GENOTYPING
Yaroslav Pirko, Anastasiia Rabokon, Anastasiia Postovoitova, Lubov Kalafat,
Yuliia Bilonozhko, Yaroslav Blume

Institute of Food Biotechnology and Genomics of the NAS of Ukraine
Kyiv, Ukraine; E-mail.: yarvp1@gmail.com

Camelina sativa (L.) Crantz. is an ancient oilseed crop, the most promising current
source of biofuels. In this regard, a number of different studies of the cultivated species C.
sativa are being conducted, and its new forms and varieties are being created and introduced.
It is often difficult to assess its genetic diversity level, therefore the selection of the most
effective molecular-genetic markers for the study of Camelina Crantz remains relevant. For
this purpose, we carried out molecular-genetic differentiation and genotyping of Ukrainian
varieties and forms of C. sativa using the intron length polymorphism of ß-tubulin and actin
genes methods.
This approach is based on simple EPIC-PCR. The universality and simplicity of these
methods in the molecular-genetic analysis of plants have already been proved in our earlier
researches. Genomic DNA was extracted from fresh leaf tissue and after PCR amplification,
performed with the 4 different pairs degenerated primers, the products were analyzed on a
non-denaturing acrylamide gel. The results of the 1st intron length polymorphism of the ß-
tubulin gene analysis showed that the bands were in the range of 295–3200 bp. Wherein, only
7 bands are polymorphic and observed only in a part of the samples, i.e, all of the samples
could not be differentiated by this marker. The results of the 1st intron length polymorphism
of the actin gene analysis demonstrated that bands are formed in the range of 370–1000 bp, 8
bands are polymorphic. It was possible to differentiate a part of the samples (FEORZhYaF-3,
Euro-12, FEORZhYaF-4, FEORZhYaF-D), which did not differ according to the analysis of the
previous marker. In order to conduct more accurate profiling of samples, an analysis of the
2nd intron length was involved. Using the 2nd intron length polymorphism of the ß-tubulin
gene, bands were within 350–1990 bp, 6 bands were polymorphic. And in this case, most of
the samples were clearly differentiated from each other, having their own unique DNA profile.
All the investigated samples were similar for the 2nd intron length polymorphism of the actin
gene. In all cases, the large number of bands was formed, that confirms the fact of polyploidy.
Thus, we found a low level of intervarietal polymorphism of C. sativa. This may indicate
a high degree of genetic similarity, which is consistent with data obtained using AFLP, RAPD
and SSR markers. Also, we determined that the 2nd intron length polymorphism of the ß-
tubulin gene and the 1st intron length polymorphism of the actin gene are the most effective
methods in the case of C. sativa profiling, and can be useful in the selection process, for
example, in selecting parental pairs or genotyping new varieties.

Keywords: Camelina sativa, molecular-genetic markers, ß-tubulin and actin genes, introns.
Acknowledgments
This work was particularly supported by the project of National Academy of Sciences of Ukraine
“Creating molecular and genetic markers for differentiation genotypes of plants by studying
polymorphisms of introns of their cytoskeleton proteins” (0115U005025) and “Differentiation of
different genotypes and varieties of plants by evaluating the introns length polymorphism of actin
genes” (0118U006867).

4 International Scientific Conference Agrobiodiversity Nutrition, Health and Quality of Human and Bees Life |125
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September 11–13, 2019