Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2018, Vol. 53, ¹ 3, p. 499-510.
(how to cite this article)

doi: 10.15389/agrobiology.2018.3.499eng

UDC 633.11:577.21

Acknowledgments:
The wheat-wheatgrass hybrids by courtesy of Dr V.I. Belov and Dr Upelniek (Tsitsin State Botanic Garden RAS, Moscow), and the bread wheat lines with added Thinopyrum elongatum chromosomes (created by Dr Jan Dvorak, UC Davis, USA) by courtesy of Dr J. Raupp (Kansas State University, USA)
Supported financially by Russian Science Foundation, Agreement ¹ 16-16-00097 of January 27, 2016

 

CLONNING OF DREB1 GENE IN WHEAT WILD RELATIVES AND
DEVELOPMENT OF A DNA MARKER FOR ITS MONITORING IN
WHEAT BACKGROUND

A.A. Pochtovyy1, 2, P.Yu. Kroupin1, 3, M.G. Divashuk1, 3,
A.A. Kocheshkova1, P.A. Sokolov1, G.I. Karlov1, 3

1Timiryazev Russian State Agrarian University—Moscow Agrarian Academy, Center for Molecular Biotechnology, 49, ul. Timiryazevskaya, Moscow, 127550 Russia, e-mail a.pochtovyy@gmail.com, pavel-krupin@yandex.ru, divashuk@gmail.com, alina.korotaeva@gmail.com, pav2395147@yandex.ru;
2All-Russian Research Institute of Agricultural Biotechnology, Federal Agency of Scientific Organizations, 42, ul. Timiryazevskaya, Moscow, 127550 Russia;
3Lomonosov Moscow State University, Faculty of Biology, 1-12 Leninskie Gory, Moscow, 119991 Russia, e-mail karlovg@gmail.com (✉ corresponding author)

ORCID:
Pochtovyy A.A. orcid.org/0000-0003-1107-9351
Kroupin P.Yu. orcid.org/0000-0001-6858-3941
Divashuk M.G. orcid.org/0000-0001-6221-3659
Kocheshkova A.A. orcid.org/0000-0003-1924-6708
Sokolov P.A. orcid.org/0000-0002-9301-8175
Karlov G.I. orcid.org/0000-0002-9016-103X

Received October 31, 2017

 

The DREB gene encodes the transcription factor DREB involved in the response of the plant to drought, salinity and heat. The DREB transcription factors induce the expression of multiple genes linked by signal transmission, abscisic acid-dependent and independent, in response to abiotic stress. Many wild species have evolved under extreme environmental conditions (drought, salinity), so they can serve as sources of new genetic variants of DREB in breeding wheat for stress resistance. The study of DREB orthologous genes in wild relatives of wheat will permit to expand the set of genes in its breeding improvement using wide hybridization, and the design and application of molecular markers will facilitate transfer of these genes into a genome of bread wheat. Among the diversity of genes encoding DREB proteins, DREB1 is of the greatest interest due to its involvement in control of plant resistance to various abiotic factors, such as drought, salinity, low temperatures. We were the first to study the DREB1 orthologs in members of the genera Thinopyrum, Dasypyrum and Pseudoroegneria. Using primers designed on the basis of DREB1 conserved regions we amplified fragments of the DREB1 orthologs in Thinopyrum intermedium, Th. ponticum, Th. bessarabicum, Dasypyrum villosum, Pseudoroegneria spicata, and P. stipifolia. The obtained PCR products were cloned and sequenced. As a result, 30 unique sequences were shown to be highly homologous (92-98 %) to the TaDREB genes of bread wheat. Between the sequences, we identified multiple single-nucleotide polymorphisms (SNPs) and several large insertions/deletions. The resulting DNA sequences were translated in silico into hypothetical amino acid sequences. All nucleotide sequences found by us are capable of encoding a complete protein that has a DNA-binding domain specific for DREB AP2. Comparison of the amino acid sequences of the AP2 DNA-binding domain in the studied samples showed the presence of polymorphisms for individual amino acids. In all hypothetical amino acid sequences, except for one the sequence described, amino acids conserved for the DREB AP2-domain of cereals were found. We developed the CAPS marker P18_FokI, which in most cases can differentiate the DREB1 orthologs between wheat and wild relatives due to the presence of polymorphisms in the restriction sites, the fragment amplified from the genome of bread wheat has a size of about 570 bp. A DREB1 ortholog was localized in the homeological group 3 of Th. elongatum (3Je) using P18_FokI and a series of addition bread wheat lines with Th. elongatum chromosomes. Analysis of 10 wheat-wheatgrass hybrids revealed the presence of both TaDREB bread wheat and the DREB1 ortholog in all analyzed accessions. In this case, the wheat-type fragment was absent in bread wheat with a substituted chromosome 6J (6D), which also serves as a proof of localization of the DREB1 ortholog on the chromosome of homeological group 3. Thus, the CAPS marker P18_FokI developed by us helps to effectively transfer the DREB1 gene from the wild cereals to the genome of bread wheat, so that we can study the effect of the alien DREB1 gene on the resistance of bread wheat to drought, salinity, low temperatures, and, farther, to create valuable breeding forms using MAS.

Keywords: bread wheat, Triticum, Thinopyrum, Pseudoroegneria, Dasypyrum, resistance genes, orthologous genes, drought, salinity, DREB1, DREB, TADREB1, polymerase chain reaction, molecular markers, sequencing.

 

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