ISSN 2219-3782. Ôàêòîðè åêñïåðèìåíòàëüíî¿ åâîëþö³¿ îðãàí³çì³â. 2015. Òîì 16 83 Many genes for resistance to biotic and abiot- ic stresses have been transferred to the genomes of modern wheat cultivars from wild relatives of wheat [1]. Amblyopyrum muticum (Boiss) Eig (Aegilops mutica Boiss) is one of diploid wild relatives of wheat which is resistant to fungal diseases, and until recently was not much used for introgressive hybrid- ization [2, 3]. Aurotica is a genome substitution am- phidiploid which combines in its genome (AABBTT) tetracomponent (AABB) from common wheat win- ter cultivar Aurora, and T genome of Aegilops muti- ca [4]. During a 20-year period of monitoring, Au- rotica demonstrated resistance to powdery mildew and rust, and additionally was characterized by high- er winter hardiness. Using genome mixing method [5], wheat introgressive lines are being developed, which contain different amounts of T genome chro- matin in their genomes, and are derived from the in- itial Aurora x Aurotica cross. Aurotica (AABBTT) is characterized by higher level of winter tolerance compared to Aurora cultivar (AABBDD), and this is supposed to be determined by genes of T genome. Freezing tolerance and winter hardiness in wheat is controlled by genes on chromosomes of homeolog- ical group 5 [6], for this reason analyzing the devel- oped lines, fi rst of all we tried to identify those con- taining introgressions in chromosomes of this group. Microsatellites (SSRs) can be used as markers for identifi cation of alien chromatin in wheat genome. Microsatellite loci specifi c to chromosomes 5A, 5B, and 5D were used to identify chromatin from T ge- nome in the genomes introgressive lines. Materials and methods Plant material: common wheat cultivar Au- rora, genome substitution amphidiploid Aurotica, 234 42-chromosome F 5 plants from crossing Au- rotica and Aurora. DNA was extracted from leaves using CTAB buffer. PCR was done with primers to microsatellite loci specifi c to the chromosomes UDC 631.523:581.13 IEFIMENKO T. S.1, FEDAK G.2, ANTONYUK M. Z.1, TERNOVSKA T. K.1 1 National University of «Kyiv-Mohyla Academy», Ukraine, 04070, Kyiv, Skovorody str., 2, e-mail: centaureinae@gmail.com 2 Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Center, Ottawa, Ontario INTROGRESSIVE WHEAT LINES TRITICUM AESTIVUM/AMBLYOPYRUM MUTICUM AS TO STRUCTURE OF HOMOEOLOGOUS GROUP 5 CHROMOSOMES © IEFIMENKO T. S., FEDAK G., ANTONYUK M. Z., TERNOVSKA T. K. of homeological group 5 [7, 9, 10]. Reaction mix- ture contained 250 nM of each primer, 50 ng of DNA, 0,2 mM of each dNTP, 1,5 mM MgCl 2 , 1,2 u Taq-polymerase (Fermentas). Conditions of ampli- fi cation were used as recommended by primers de- veloper. Amplifi cation products were separated in denaturating PAAG. Results and discussion Hexaploids Aurora and Aurotica differ from each other by one subgenome: D genome in Aurora and T genome in Aurotica. Tetraploid component AABB in these forms must be identical. For this reason, am- plifi cation spectra for chromosomes 5A and 5B are also supposed to be identical for Aurora and Aurotica, which was true for 46 loci on chromosomes 5A and 22 loci on chromosome 5B from 62 and 29 analyz- ed, respectively. Primers specifi c to 5D chromosome must not amplify any product with DNA of Aurotica, because it does not contain subgenome D. Neverthe- less, amplifi cation product was absent only for two loci from 24 analyzed. This phenomenon could be explained by the transferability of microsatellite loci specifi city among chromosomes of the same homeo- logical group of Triticinae. This fact has been known from the beginning of use of microsatellite loci for mapping and comparison of Triticinae genomes [11]. Transferability of microsatellite loci is confi rmed on our material: 8 pairs of primers from 102 analyzed were specifi c to two chromosomes of homeological group 5, and 3 pairs of primers were shown to be spe- cifi c to all three homeological chromosomes. Accord- ing to our results, transferability among D and T ge- nomes is even higher then among other subgenomes of wheat. This could be considered as an evidence for D and T genomes homology, especially taking into account conjugation of the chromosomes from these genomes [4, 12]. Because of the transferability of the microsatel- lite loci among chromosomes of homeological group 84 ISSN 2219-3782. Ôàêòîðè åêñïåðèìåíòàëüíî¿ åâîëþö³¿ îðãàí³çì³â. 2015. Òîì 16 Iefimenko T. S., Fedak G., Antonyuk M. Z., Ternovska T. K. 5, including 5T chromosome, DNA of Aurora and Aurotica can produce identical spectra of amplifi ca- tion products with primers to loci specifi c to more than one chromosome (fi g. 1). Fig. 1. Localization of analyzed microsatellite loci on 5D chromosome; distance from the distal part of the short arm of chromosome is shown in map units [7, 9, 10] If SSR loci from T genome have alleles differ- ing from Aurora, then specifi c components appear in spectra of Aurotica. It was true for microsatellite loci Xcfa2151 (5A/5D), Xwmc289 (B, D), Xgdm68 (5A/5B/5D) та Xgwm293 (A, B, D). When primers to the loci Xwmc233, Xcfd18, Xcfd8, Xgpw5098, Xgwm292, Xcfd29, Xcfd183, Xwmc765, Xwmc443, Xgwm654 produce identical spectra with DNA of Aurora and Aurotica, this indicates that these loci are specifi c not only to the genome D, but also to the genome T, and alleles at these loci in Aurora and Aurotica are identical. The same explanation is true for the loci Xcfa2104 (A, D), Xcfa2185 (A, D), Xbarc316 (A, D), Xbarc232 (A, B, D). For the identifi cation of T genome chromo- somes in the genomes of introgressive lines only 9 microsatellite loci are appropriate: Xcfd189, Xcfd102, Xgwm190, Xgwm272, Xgwm182, Xcfa2141 (5A/5D) Xgdm68 (A, B, D), Xgwm293 (A, B, D) Xwmc289 (B, D). Aurotica has specifi c product of amplifi ca- tion with the primers to these loci. The fact that four of these 9 loci produce amplifi cation product with A and B genomes, could be theoretically ignored, be- cause this part of the genomes of Aurora and Aurot- ica must be identical and differences in spectra are supposed to be caused by the presence of different al- leles of these microsatellite loci in D and T genomes. Microsatellite locus Xcfd57 does not produce ampli- fi cation product with DNA of Aurotica, which could be caused by the absence (or rearrangement) of the corresponding region of 5D chromosome. This locus provides no evidence about the presence of chromo- some segment from T genome. As for 5A and 5B chromosomes, difference in spectra of amplifi cation products between Au- rora and Aurotica was observed for 12 loci on the chromosome 5A (32 % from the studied loci) and for 2 loci on the chromosome 5B (14 % of studied loci). Differences between Aurora and Aurotica for microsatellite loci Xwmc752, Xwmc150, Xcfd40, Xbarc117, Xwmc805, Xbarc1, Xgwm186, Xbarc142, Xgwm179, Xcfd39, Xgwm291, Xcfd47 (chromosome 5А), and Xgwm544, Xcfd7, Xwmc160 (chromosome 5В), can be caused by one of the three factors. First, while extracting AABB tetracomponent from the AABBDD genome of common wheat, which was done by crossing Aurora cultivar with durum wheat and six backcrosses of pentaploid hybrids with Au- rora, recombination between chromosomes of A and B genomes of common and durum wheat occurred [13]. That could be the reason for partly disrupted identity between tetra-Aurora and Aurora AB ge- nomes. Second, information about transferability of studied microsatellite loci among chromosomes of one homeological group was obtained not on Auro- ra, but other wheat genotypes [7, 9, 10]. It is pos- sible, that in Aurora genome these loci are specif- ic also to 5D chromosome, and it is also possible, that they are as well specifi c to chromosome 5T of Aurotica. Third, during last decade many works, in- cluding those on Triticinae, demonstrated that am- phidiploidization process (development of Aurotica included amphidiploidization) is accompanied by numerous, and possibly directed, rearrangements in the genome, and these rearrangements include changes in microsatellite loci [14]. For screening of 234 42-chromosome F 5 plants from crossing Aurora x Aurotica 7 SSR loci with diagnostic value for identifi cation of substitution of microsatellite locus characteristic to 5D chromo- some to that specifi c to 5T were used (fi g. 1). Aurora x Aurotica hybrid had AABBDT genome structure. Conjugation between D and T chromosomes was demonstrated before, although some univalents were also present in metaphase [4, 12]. This is confi rmed by variation of chromosome numbers from 33 to 46 among F 2 offsprings of Aurora x Aurotica hybrid, which occurs because of irregular segregation of chromosomes of D and T genomes to the poles of meiocytes. Only fertile plants participated in forma- ISSN 2219-3782. Ôàêòîðè åêñïåðèìåíòàëüíî¿ åâîëþö³¿ îðãàí³çì³â. 2015. Òîì 16 85 Introgressive wheat lines Triticum Aestivum/Amblyopyrum muticum as to structure of homoeologous group 5 chromosomes tion of next hybrid generation, which caused nar- rowing of the variation of chromosome numbers, and in F 5 generation offsprings of only 15 F 2 plants were present. In generations from F 2 to F 5 plants with chromosome numbers near 42 were fertile; they were formed from gametes with almost normal chro- mosome constitution. For this reason it is credibly that among 15 F 2 plants whose F 5 offsprings were analyzed, ¾ of plants have at least one chromosome 5T (11 plants from 15), and ¼ have only 5D chro- mosomes (4 plants from 15). Comparison of ampli- fi cation products spectra of F 5 plants demonstrated that 5T chromosome was present in the genomes of 13 F 2 plants, and it was absent in two plants, which corresponded to the expected chromosome numbers (according to the Fisher’s exact test, P = 0,651). If chromosomes 5D and 5T conjugate, recom- bination can occur between them, and among off- springs of successive generations not only plants with intact 5T chromosome, but also plants with recombinant 5D/5T chromosome may be present. According to the obtained results, it was possible to make hypothesis about chromosome constitution of plants from generations F 2 –F 4 as to the presence of 5T chromosome: was it mono- or disomic substitu- tion 5D/5T. Additionally, it was possible to identify plants with recombinant 5D/5T chromosomes. Evi- dence that a plant from F 2 generation had a pair of nonrecombinant chromosomes could be represented only as presence of the same (nonrecombinant) chro- mosome constitution in F 5 generation, and therefore in all in-between generations, which were not ana- lyzed. Only one such plant was identifi ed among F 2 generation. Although that plant could have another chromosome structure, it could not be demonstrat- ed from analysis of the existent offsprings. Other 12 plants must have had two intact chromosomes, 5D or 5T, or one or both of them were recombinant and contained alleles of microsatellite loci charac- teristic to both chromosomes. These two cases could not be distinguished basing on our results: if F 2 plant had two intact chromosomes 5D and 5T, recombina- tion between them could have occurred during the formation of gametes of any following generation, however only F 5 generation was analyzed. Among the plants of F 3 generation only 6 plants could possi- bly have nonrecombinant 5T chromosome in the ge- nome. One of them could have disomic substitution 5D/5T, because all the analyzed F 5 offsprings of this plant were characterized by this chromosome con- stitution. Five other F 3 plants possibly had one intact 5T chromosome, and one chromosome with rear- rangements and insignifi cant (three plants) or sig- nifi cant (one plant) inclusion of fragments from 5T chromosome. Among the offsprings of the F 3 plants, in F 5 generation 21 plants which can be considered disomic substitution lines 5D/5T were identifi ed us- ing microsatellite analysis. All the rest plants from F 3 and F 4 generations contained in their genomes recombinant chromosomes 5D/5T, which combined fragments of these chromosomes in different com- binations. Numerous 5D/5T recombinant chromosomes were observed among F 5 plants, and recombination events could occur in any generation beginning from F 2 . This confi rms hypothesis [12] about homology of D and T genomes, and indicates that T genome of Am- blyopyrum muticum belongs to the group of Triticinae genomes which can conjugate with chromosomes of at least one of three subgenomes of common wheat. These genomes are very valuable for introgressive hybridization as they give the possibility to obtain in- trogression of small fragments of alien chromosomes into homeological chromosome of wheat through re- combination process (without application of specifi c methods). It is important to obtain translocations from alien chromosomes, which may carry useful genes, in very small amount of alien chromatin. Identifi ed plants with 5D/5T recombinant chromosomes are promising material for future work on introgressive hybridization and genetic analysis. On the other hand, recombination between D and T chromosomes makes more diffi cult identifi ca- tion of alien disomic substitution lines. When D chro- mosomes do not conjugate with chromosomes from alien genome, it is possible to use only 1–2 biochem- ical markers on chromosome combined with analy- sis of univalent numbers in metaphase I of hybrids among recipient genotype (Aurora) and 42-chromo- some introgressive line for identifi cation of substitu- tions of D genome chromosomes by chromosomes of genomes S, Ssh, and U of three wild species: Aegilops speltoides, Ae. sharonensis, and Ae. umbellulata, re- spectively. However, when chromosomes of donor (T) and recipient (D) genomes conjugate and form re- combinant chromosomes with frequency much higher that frequency of preservation of intact chromosomes in generations, identifi cation of such chromosomes requires screening of the plant material with several marker loci per chromosome. Markers should cover both chromosome arms, and preferably on distal and proximal regions from centromere. In electrophoretic spectra of some plants ap- peared such component (amplifi cation product) that differed from both Aurora and Aurotica components, and it was designated as «new» one. Appearance of 86 ISSN 2219-3782. Ôàêòîðè åêñïåðèìåíòàëüíî¿ åâîëþö³¿ îðãàí³çì³â. 2015. Òîì 16 Iefimenko T. S., Fedak G., Antonyuk M. Z., Ternovska T. K. new alleles in microsatellite loci can be explained by mutations, as their frequency is higher in genome re- gions with repeats (such as SSR loci). From 7 studied microsatellite loci, such «new» alleles were absent only for two loci — Xgdm68 (A, B, D) and Xgwm182. New alleles appeared not only on chromosomes D and T, but also on chromosomes of A and B subgenomes. Conclusions Comparison of amplifi cation products spectra produced with DNA of Aurora and Aurotica with primers to microsatellite loci specifi c from one to three chromosomes of homeological group 5, gave the possibility to identify loci which are diagnosti- cally valuable for identifi cation of plants with sub- stitution 5D/5T and recombination 5D/5T among 42-chromosome offsprings of F 5 hybrid from Aurora x Aurotica cross. Among total 234 plants 24 plants with disomic substitution 5D/5T were identifi ed. Numerous plants carry recombinant chromosome 5D/5T. Recombination event could have occurred in any generation beginning from F 2 . Microsatellite analysis of introgressive material has some limita- tions because of high mutation rate in SSR loci, and potential transferability of these loci on more than one chromosome of the same homeological group. Plants with recombinant chromosomes 5D/5T are promising material for future research. REFERENCES 1. Kazi A. G. 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The homoeology of Aegilops mutica chromosomes. // Canadian Journal of Genetics and Cytology. — 1968. — 10 (3). — P. 620–626. 13. Терновская Т. К., Жиров Е. Г. Выделение тетраплоидного компонента ААВВ из мягкой пшеницы сорта Сара- товская 29 // Докл. ВАСХНИЛ. — 1979. — № 3. — С. 8–10. 14. Feldman M., Levy A. A. Allopolyploidy — a shaping force in the evolution of wheat genomes // Cytogenet Genome Res. — 2005. — 109. — P. 250–258. IEFIMENKO T.S.1, FEDAK G.2, ANTONYUK M.Z.1, TERNOVSKA T.K.1 1 National University of «Kyiv-Mohyla Academy», Ukraine, 04070, Kyiv, Skovorody str., 2, e-mail: centaureinae@gmail.com 2 Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Center, Ottawa, Ontario INTROGRESSIVE WHEAT LINES TRITICUM AESTIVUM / AMBLYOPYRUM MUTICUM AS TO STRUC- TURE OF HOMOEOLOGOUS GROUP 5 CHROMOSOMES Aims. Using SSR loci specifi c to homeological group 5 chromosomes, identify T genome introgressions in the genomes of F 5 wheat plants from crossing Aurora x Aurotica. Methods. DNA extraction using CTAB method, PCR with primers to SSR loci, electrophoresis in PAAG, silver staining. Results. Comparative microsatellite analysis of Aurora and Aurotica identifi ed 9 SSR loci specifi c to D genome which produced differing amplicons with Aurotica DNA. These loci were used for screen- ing (Aurora x Aurotica) F 5 progeny (234 plants); 24 plants with disomic substitutions 5D/5T were detected, and a signifi cant number of plants containing recombinant chromosome 5D/5T. The recombination event could occur in any generation from F 2 . Conclusions. Microsatellite loci could be used for identifi cation of introgressions from Aegilops mutica in wheat genome (although with some limitations). Identifi ed plants with recombinant 5D/5Т chromosomes are perspective for future work. Keywords: introgressive hybridization, Amblyopyrum muticum, microsatellite analysis, SSR loci, alien-substitution lines, recombinant lines.