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MICROSATELLITE LOCI POLYMORPHISM IN MIGUSCHOVA WHEAT AND COMMON WHEAT CULTIVARS

Fusarium head blight is one of the most widespread and dangerous wheat diseases worldwide. Resistance to Fusarium is controlled by some main genes from different Triticinae genomes; however, common wheat has few such resistance genes. Miguschova wheat (Triticum miguschovae Zhir.) with AbAbGGDD genome is characterized by the genotype resistant to Fusarium head blight. In order to effectively use it as a source of introgressions to common wheat genome, molecular genetic polymorphisms should be identified, which could later be used for identification of Miguschova wheat introgressions in the common wheat genome. Microsatellite PCR analysis using primers to SSR loci with a known chromosome localization for common wheat identified 14 highly informative loci with specific to Miguschova wheat amplicons, localized on chromosomes of 6 homoeological groups. Seven other SSR loci were identified to have a limited informative value, as DNA of Miguschova wheat did not form any specific PCR product with corresponding primers (null allele). The informative value of those loci was limited to differentiation of wheat cultivars.

Fusarium head blight is one of the most dangerous and widespread diseases of cereals and wheat in particular. Characteristic feature of this disease is that fungi from the Fusarium genus not only harm plants, but also a number of mycotoxins accumulate in grain. Therefore, on contrast to other phytopathogenic lesions, the harvest becomes unusable. No fully resistant to Fusarium head blight wheat cultivars have been developed up to know [1][2][3]. Resistance to the disease is controlled by a number of main genes with different chromosome localization; and wheat wild relatives are important sources of new resistance genes to Fusarium head blight. Migusсhova wheat (Triticum miguschovae Zhir., A b A b GGDD genome) is resistant to Fusarium head blight according to G. Fedak [4]. The use of this species for development of introgressive lines with common wheat as a recipient genome (T. aestivum L, AABBDD genome) is promising for enrichment of common wheat with genes for resistance to Fusarium head blight. The process of introgressive lines development includes as a required element screening of hybrid progeny with the use of molecular genetic markers for identification of alien chromatin in their genomes. Use of microsatellite (SSR) loci for identification of introgressions in wheat is widespread and productive direction for screening introgressive derivatives of distant crosses [5][6][7][8]. Search for polymorphism of chromosome specific SSR loci in components of initial cross (common wheat x Miguschova wheat) is a key stage in preparation for screening introgressive progeny, as on the presence of such polymorphism depends possibility of the following selection of those hybrids that contain introgressions in their genomes. The article offers the results of a comparative microsatellite analysis of genomes of Miguschova wheat and common wheat cultivars, and these results could be the basis for selection of cross combinations perspective for the future work with their derivatives.

Materials and methods
Wheat genotypes analyzed in the study: DNA was extracted from leaves using buffer containing CTAB. DNA was amplified with primers to microsatellite loci in PCR with conditions according to the originators of primers. DNA was extracted from individual plants. Chromosome and chromosome arm specificity of studied SSR loci are shown in the tables 1-7. Amplification products were electrophoretically separated in 8 % PAAG with 6M carbamide.

Results and discussion
Search for polymorphism between Miguschova wheat and common wheat cultivars were conducted through comparison of electrophoretic spectra of amplification products obtained using primers to microsatellite loci with chromosome specificity for common wheat. Polymorphism was identified as different electrophoretic mobility of spectra components (+/+), or as presence/absence of particular component in the compared spectra (-/+). For Xgpw1143, Xcfd92, Xgwm337, Xgwm106 no polymorphism was identified for mobility of spectra components (table 1). Differences in spectra were identified only for three SSR loci ( fig. 1), and in all cases polymorphism was identified as different weight (mobility) of amplification products obtained with DNA of Miguschova and common wheat.
Out of the six studied SSR loci specific to the chromosomes of homoeological group 2 (table 2), three loci were polymorphic ( fig. 2): Xbarc124-2A and Xgwm122-2A having alleles of different length, and locus Xgwm311, for which amplification products were obtained only with DNA of Miguschova wheat, and no amplification products were obtained for studied common wheat cultivars. Locus Xgwm261-2D was monomorphic between studied genotypes, and primers to loci Xgwm539-2D and Xgwm304-2A produced no amplification products.
Only three microsatellite loci were studied for chromosomes of homoeological group 3, and for all these loci polymorphisms of "+/-" type were identified: presence of amplicons in spectra of common wheat cultivars and absence of amplification products with DNA of Miguschova wheat (table 3). Informative value of such polymorphism is unreliable and limited for analysis of introgressive plant material. Three SSR loci specific to chromosome of homoeological group 4 of wheat were monomorphic for studied genotypes, and two loci demonstrated polymorphism ( fig. 3, table 4). With primers to locus Xwmc285 DNA of Miguschova wheat produced three amplicons whereas common wheat cultivars produced two amplicons; informative value of this locus is high. With primers to locus Xgwm194 DNA of common wheat produced four amplicons, and DNA of Miguschova wheat produced no amplification products; informative value of this locus is limited.  Table 3. Characteristics of the primers to microsatellite loci on chromosomes of the homoeological group 3

Presence of amplification product Polymorphism presence and type Miguschova wheat common wheat
Xcfd55-3D For eight out of the 13 studied microsatellite loci specific to chromosomes of homoeological group 5 no amplification products were obtained with DNA of both wheat species (table 5). Alleles of SSR loci ХCfd156-5B and Xbarc18-5D were identical for Miguschova wheat and common wheat. Only loci Xcfd86-5D, Xgwm179-5A and Xbarc230-5A were identified as having informative value, as amplification products produced with primers to these loci had different mobility in electrophoretic spectra ( fig. 4).
Among microsatellite loci specific to chromosomes of homoeological group 6 only loci Xbarc196-6D and Xcfd287-6D were identified to be informative, as their amplification products with DNA of Miguschova wheat and common wheat cultivars had different mobility on electrophoretic spectra (table 6, fig. 5). SSR loci Xcfd132-6D and Xcfd76-6D had limited informative value, as DNA of Miguschova wheat did not produce any amplification products with primers to these loci. Alleles of Xbarc96-6D locus were monomorphic.
Comparing the components of spectra obtained after electrophoresis of samples amplified with primers to SSR loci Xbarc53, Xwmc506, Xbarc111, Xgwm44, Xcfd69 and Xbarc154, it was demonstrated that all studied genotypes produced identical components, therefore, no polymorphism was identified. Polymorphism was identified for SSR loci Xcfd66 and Xbarc172 (table 7, fig. 6). For locus Xbarc172 Miguschova wheat DNA formed amplicons with different electrophoretic mobility    6). Three other components were present in all the studied samples.

Conclusions
Polymorphism between Migusсhova wheat and common wheat was studied using molecular genetic markers specific to chromosomes of different homoeological groups of wheat. Among 52 studied microsatellite loci with known localization on chromosomes of seven wheat homoeological groups [9][10][11][12], 14 loci were identified to have high informative value: 3, 3, 0, 1, 3, 2, 2, according to chromosomes of homoeological groups from 1 to 7. Seven other SRR loci were identified to have limited informative value, because DNA of Miguschova wheat did not produce specific amplification products with primers to these loci (null allele). Thereby, according to microsatellite analysis not all chromosomes of Miguschova wheat could be identified as substituted in the introgressive derivatives from its cross with common wheat cultivars. However, if in addition to microsatellite markers, we compare results of protein polymorphism study between Miguschova wheat and common wheat cultivars, chromosomes of all homoeological groups of wheat with genome A b A b GGDD could be identified [13].