# Evaluating Wheat Microsatellite Markers for the Use in Genetic Analysis ofThinopyrum, Dasypyrum, and Pseudoroegneria Species

Authors: Pavel Yu. Kroupin; Mikhail G. Divashuk; Igor A. Fesenko; Gennady I. Karlov
Journal: Dataset Papers in Biology
 (2013)

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## Abstract

A set of 42 SSRs of wheat were evaluated for their cross-amplification on the DNA ofThinopyrum ponticum, Thinopyrum intermedium, Thinopyrum elongatum, Thinopyrum bessarabicum, Pseudoroegneria stipifolia, and Dasypyrum villosum. The number of the wheat SSR markers that amplified DNA fragments with determined size for Th. ponticum was 33 (78.6%); for Th. intermedium, 28 (66.7%); for Th. elongatum, 24 (57.1%); for Th. bessarabicum, 24 (57.1%); for P. stipifolia, 26 (69.1%); and for D. villosum, 29 (69.0%). Twenty-four primer pairs of wheat SSR markers were successfully amplified from all investigated species. The dataset can be used for phylogenetic studies of wild relatives of wheat, for the estimation of their diversity, and for the introgression of agronomically valuable genes into wheat genome.


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## Body

## 1. Introduction

Wheat is a major food source for most world population. However, its cultivation is strictly limited by such challenges as pests, diseases, droughts, extreme temperatures, and others. In order to manage sustainable wheat production, it is necessary to develop new cultivars of wheat resistant and tolerant to adverse environmental factors. The genetic diversity of wheat is restricted due to the bottleneck effect which occurred during domestication and, on the other hand, due to the limited diversity of wheat germplasm used in the breeding programs [1, 2]. The genetic variability of cultivated wheat can be improved by wide hybridization involving wild relative species. One of the important gene pools is different species of Thinopyrum, Dasypyrum, and Pseudoroegneria genera [3, 4]. These genetic resources can be efficiently used in wheat breeding by application of molecular microsatellite markers. Microsatellites also known as Simple Sequence Repeats (SSRs) are tandem repeats of two to six nucleotide DNA motifs randomly distributed all over genome. They have been successfully used as DNA markers to estimate genetic diversity, genome mapping, and marker-assisted selection for agronomically important traits [5]. SSR markers are a powerful tool for the investigation of cereal genomes because of the high level of polymorphism they reveal [6]. In addition, SSRs can be used in wide hybridization of wheat to monitor and map desirable alien genes in segregating populations as they are locus specific and inherited in codominant manner [7].A large number of the primer sequences of wheat SSRs are publicly available. However, the development of new SSR markersde novo for wild relatives of wheat is a time- and cost-consuming process. The flanking regions of microsatellites show sufficient homology between closely related species. Thus, the primers of wheat SSR markers can be used in the analysis of other Poaceae species. This is called “transferability” and it has been successfully used for cultivated (barley, oats, and rye) [8] and wild (Elymus,Aegilops) species [9, 10]. The goal of our study was to test a set of 42 microsatellite markers of wheat for their ability to cross-amplify DNA from the accessions of Th. intermedium, Th. elongatum, Th. ponticum, Th. bessarabicum, P. stipifolia, and D. villosum.


## 2. Methodology

To get the plant materials, the following accessions were ordered from Germplasm Research International Network:Th. ponticum (PI 547312), Th. intermedium (PI 401200), Th. bessarabicum (PI 531711), Th. elongatum (PI 401007) P. stipifolia (W6 21759), and D. villosum (W6 21717). Two plants of each accession were analyzed. DNA was extracted from etiolated seedling by the standard CTAB method.Polymerase chain reaction (PCR) and fragment analysis were performed as follows. PCR was performed in final volume of 25μL in thermal cycler using the conditions recommended at GrainGenes 2.0 [11] according to the protocol by Schuelke [12]. The first round of PCR was conducted using reverse (0.16 pmol/μL) and forward primer with M13 tail (0.04 pmol/μL). During the second round, the fluorescent-labeled M13 primer was incorporated into the PCR product at annealing temperature 53°C. Fragment analysis of PCR product was carried out using ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with GeneScan 500 LIZ size standard according to the manufacturer's recommendations. The sizes of fragments were analyzed using Peak Scanner Software v1.0 (Applied Biosystems, Foster City, CA, USA).


## 3. Dataset Description

The dataset associated with this Dataset Paper consists of one item which is described as follows.1
Size of the microsatellite fragments (base pair (bp)) amplified on DNA ofTh. intermedium, Th. elongatum, P. stipifolia, Th. bessarabicum, Th. ponticum, and D. villosum. A total of 42 SSR markers of wheat were evaluated on their cross-amplification on DNA of Th. intermedium, Th. elongatum, Th. ponticum, Th. bessarabicum, P. stipifolia, and D. villosum. For each accession, two plants were analyzed. The sizes of the peaks are obtained by means of the fragment analysis. The number of the wheat SSR markers that amplified DNA fragments with determined size for Th. ponticum was 33 (78.6%); for Th. intermedium, 28 (66.7%); for Th. elongatum, 24 (57.1%); for Th. bessarabicum, 24 (57.1%); for P. stipifolia, 26 (69.1%); and for D. villosum, 29 (69.0%). Thus, the transferability of the SSR markers to the genomes of the investigated species is comparable to that of oat, rye [8], Aegilops [9, 13], Elymus, and Pseudoroegneria [10]. Twenty-four primer pairs of wheat SSR markers were successfully amplified from all investigated species. The absence of amplification may result from either null allele in a locus or significant differences between wheat and studied species in the flanking regions where primers should anneal [14]. The transferred SSRs can be linked to useful genes or QTLs as among selected markers there are some previously used for the mapping and detection of resistance genes of Th. intermedium (Xwmc221, Xwmc121, and Xcfd68) [15] and Th. elongatum (Xgwm325, Xgwm179, and Xgwm335) [16, 17] and others are linked to QTLs of valuable traits on different wheat chromosomes [18]. The size shown in the table is the sum of the real size of a fragment and 25 bp of M13 tail of forward primer. The localization on wheat chromosomes is shown according to Graingenes 2.0 database [10]. If no peaks are detected, they are shown as “0” size. Where the variants between plant genotypes are observed, they are shown through slash “/”. If a group of peaks is found for one plant and it differs from the group of another plant, the sizes of peaks are shown in brackets. Cases when it is difficult to determine major peaks among multiple peaks are designated “ND” (not determined).
43
8
0
SSR Marker
Localization in Wheat Chromosome
Size of the Amplified Fragments ofTh. intermedium
Size of the Amplified Fragments ofTh. elongatum
Size of the Amplified Fragments ofP. stipifolia
Size of the Amplified Fragments ofTh. bessarabicum
Size of the Amplified Fragments ofTh. ponticum
Size of the Amplified Fragments ofD. villosum


## 4. Concluding Remarks

In conclusion, it has been shown that most of 42 SSRs are applicable for the investigation of genomes ofTh. ponticum, Th. intermedium, Th. elongatum, Th. bessarabicum, P. stipifolia, and D. villosum. The polymorphism in SSR fragment size between different analyzed species has been shown. The transferred SSR markers can be used in assaying the genetic variability of these wild species and establishment of phylogenetic relationships between them. In addition, they can be used in wide hybridization of wheat to monitor and map alien introgressions.



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*Source: 949637-2012-08-28.xml*