ISSN 10227954, Russian Journal of Genetics, 2015, Vol. 51, No. 12, pp. 1199–1203. © Pleiades Publishing, Inc., 2015.
Published in Russian in Genetika, 2015, Vol. 51, No. 12, pp. 1386–1390.
1199
1
INTRODUCTION
Maternally inherited genetic markers are of a par
ticular importance for the population and phylogeo
graphic studies. These are mtDNA markers in species
of the family Pinaceae [1–6], including Scots pine
(
Pinus sylvestris
L.). Previous phylogeographic studies
of this species using mtDNA [7, 8] were based only on
the two markers in the first intron of the
nad7
gene and
the second intron of the
nad1
gene, respectively. In
Russia and in the surrounding areas three haplotypes
were identified based on these markers—
a
,
c
, and
d
in
terms of [8]:
d
was distributed in Asia Minor,
c
—
mostly in Northern Europe, and
a
was ubiquitous, but
fixed in the populations east of the Volga River,
according to the updated data [9]. Thus, for the most
part of the Scots pine range mtDNA variation has not
1
The article was translated by the authors.
been studied (for example, in the Caucasus), or existed
markers were not variable (in Siberia and Trans
Baikal). The aim of the presented study was to develop
new informative mtDNA markers for Scots pine. To
do this, the noncoding regions of mtDNA were
de novo
sequenced and resequenced in trees of differ
ent geographic origin.
MATERIALS AND METHODS
We used two approaches for
de novo
sequencing to
develop PCR primers for resequencing of mtDNA
fragments in Scots pine. The first method was based on
using the inverse PCR [10] to study noncoding flank
ing regions of mitochondrial genes
coxI, matR,
and
atpA.
In this case the primers were located in the con
served parts of these genes. The second approach was
based on the next generation sequencing (NGS) and
Development of New Mitochondrial DNA Markers
in Scots Pine (
Pinus sylvestris
L.) for Population
and Phylogeographic Studies
1
V. L. Semerikov
a
, Yu. A. Putintseva
b
, N. V. Oreshkova
b
,
c
, S. A. Semerikova
a
, and K. V. Krutovsky
b
,
d
,
e
,
f
a
Institute of Plant and Animal Ecology, Ural Branch of the Russian Academy of Sciences, Ekaterinburg, 620144 Russia
email: semerikov@ipae.uran.ru
b
Genome Research and Education Center, Siberian Federal University, Krasnoyarsk, 660036 Russia
email: yuliyaputintseva@rambler.ru
c
Sukachev Institute of Forest, Siberian Branch of the Russian Academy of Sciences, Krasnoyarsk, 660036 Russia
email: oreshkova@ksc.krasn.ru
d
Georg August University of Göttingen, Göttingen, 37077 Germany
email: konstantin.krutovsky@forst.unigoettingen.de
e
Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991 Russia
email: kkrutovsky@gmail.com
f
Texas A&M University, College Station, TX 778432138, USA
email: kkrutovsky@tamu.edu
Received April 2, 2015
Abstract
—Fragments of genomic DNA of Scots pine (
Pinus sylvestris
L.) homologous to the mitochondrial
DNA (mtDNA) contigs of Norway spruce (
Picea abies
(L.) Karst.) and loblolly pine (
Pinus taeda
L.) were
resequenced in a sample of the Scots pine trees of European, Siberian, Mongolian, and Caucasian origin in
order to develop mtDNA markers. Flanking noncoding regions of some mitochondrial genes were also
investigated and resequenced. Five single nucleotide polymorphisms (SNPs) and a single minisatellite locus
were identified. Caucasian samples differed from the rest by three SNPs. Two SNPs have been linked to an
early described marker in the first intron of the
nad7
gene, and all together revealed three haplotypes in Euro
pean populations. No variable SNPs were found in the Siberian and Mongolian populations. The minisatel
lite locus contained 41 alleles across European, Siberian, and Mongolian populations, but, this locus dem
onstrated a weak population differentiation (
F
ST
= 5.8), probably due to its high mutation rate.
Keywords
: mitochondrial DNA, next generation sequencing, markers, phylogeography,
Pinus sylvestris,
Scots pine
DOI: 10.1134/S1022795415120108
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