Barbara Apolinarska

Cytogenetic analysis of Aegilops chromosomes, potentially usable in triticale (X Triticosecale Witt.) breeding

Chromosome identification using fluorescence in situ hybridization (FISH) is widely used in cytogenetic research. It is a diagnostic tool helpful in chromosome identification. It can also be used to characterize alien introgressions, when exercised in a combination with genomic in situ hybridization (GISH). This work aims to find chromosome identification of Aegilops species and Aegilops × Secale amphiploids, which can be used in cereal breeding as a source of favourable agronomic traits. Four diploid and two tetraploid Aegilops species and three Aegilops × Secale hybrids were analysed using FISH with pSc119.2, pAs1, 5S rDNA and 25S rDNA clones to differentiate the U-, M-, Ssh- and D-subgenome chromosomes of Aegilops genus. Additionally, GISH for chromosome categorization was carried out. Differences in the hybridization patterns allowed to identify all U-, M-, Ssh- and D-subgenome chromosomes. Some differences in localization of the rDNA, pSc119.2 and pAs1 sequences between analogue subgenomes in diploid and tetraploid species and Aegilops × Secale hybrids were detected. The hybridization pattern of the M and S genome was more variable than that of the U and D genome. An importance of the cytogenetic markers in plant breeding and their possible role in chromosome structure, function and evolution is discussed.

Aegilops-Secale amphiploids: chromosome categorisation, pollen viability and identification of fungal disease resistance genes.

The aim of this study was to assess the potential breeding value of goatgrass-rye amphiploids, which we are using as a “bridge” in a transfer of Aegilops chromatin (containing, e.g. leaf rust resistance genes) into triticale. We analysed the chromosomal constitution (by genomic in situ hybridisation, GISH), fertility (by pollen viability tests) and the presence of leaf rust and eyespot resistance genes (by molecular and endopeptidase assays) in a collection of 6× and 4× amphiploids originating from crosses between five Aegilops species and Secale cereale. In the five hexaploid amphiploids Aegilops kotschyi × Secale cereale (genome UUSSRR), Ae. variabilis × S. cereale (UUSSRR), Ae. biuncialis × S. cereale (UUMMRR; two lines) and Ae. ovata × S. cereale (UUMMRR), 28 Aegilops chromosomes were recognised, while in the Ae. tauschii × S. cereale amphiploid (4×; DDRR), only 14 such chromosomes were identified. In the materials, the number of rye chromosomes varied from 14 to 16. In one line of Ae. ovata × S. cereale, the U-R translocation was found. Pollen viability varied from 24.4 to 75.4%. The leaf rust resistance genes Lr22, Lr39 and Lr41 were identified in Ae. tauschii and the 4× amphiploid Ae. tauschii × S. cereale. For the first time, the leaf rust resistance gene Lr37 was found in Ae. kotschyi, Ae. ovata, Ae. biuncialis and amphiploids derived from those parental species. No eyespot resistance gene Pch1 was found in the amphiploids.

Identification of the chromosome complement and the spontaneous 1R/1V translocations in allotetraploid Secale cereale × Dasypyrum villosum hybrids through cytogenetic approaches

Genome modifications that occur at the initial interspecific hybridization event are dynamic and can be consolidated during the process of stabilization in successive generations of allopolyploids. This study identifies the number and chromosomal location of ribosomal DNA (rDNA) sites between Secale cereale, Dasypyrum villosum, and their allotetraploid S. cereale × D. villosum hybrids. For the first time, we show the advantages of FISH to reveal chromosome rearrangements in the tetraploid Secale × Dasypyrum hybrids. Based on the specific hybridization patterns of ribosomal 5S, 35S DNA and rye species-specific pSc200 DNA probes, a set of genotypes with numerous Secale/Dasypyrum translocations of 1R/1V chromosomes were identified in successive generations of allotetraploid S. cereale × D. villosum hybrids. In addition we analyse rye chromosome pairs using FISH with chromosome-specific DNA sequences on S. cereale × D. villosum hybrids.

Effective transfer of chromosomes carrying leaf rust resistance genes from Aegilops tauschii Coss. into hexaploid triticale (X Triticosecale Witt.) using Ae. tauschii × Secale cereale amphiploid forms.

This paper shows the results of effective uses of a molecular cytogenetics toolbox and molecular marker to transfer leaf rust resistance genes from Aegilops tauschii × Secale cereale (DDRR, 2n = 4x = 28) amphiploid forms to triticale cv. Bogo (AABBRR, 2n = 6x = 42). The molecular markers of resistance genes and in situ hybridization analysis of mitotic metaphase of root meristems confirmed the stable inheritance of chromosome 3D segments carrying Lr32 from the BC2F2 to the BC2F5 generation of (Ae. tauschii × S. cereale) × triticale hybrids. The chromosome pairing analysis during metaphase I of meiosis of BC2F4 and BC2F5 hybrids showed increasing regular bivalent formation of 3D chromosome pairs and decreasing number of univalents in subsequent generations. The results indicate that using amphiploid forms as a bridge between wild and cultivated forms can be a successful technology to transfer the D-genome chromatin carrying leaf rust resistance genes into triticale.

Introgression of A- and B-genome of tetraploid triticale chromatin into tetraploid rye

An improvement of rye is one of the mainstream goals of current breeding. Our study is concerned with the introduction of the tetraploid triticale (ABRR) into the 4x rye (RRRR) using classical methods of distant crossing. One hundred fifty BC1F9 hybrid plants [(4x rye × 4x triticales) × 4x rye] obtained from a backcrossing program were studied. The major aim of this work was to verify the presence of an introgressed A- and B- genome chromatin of triticale in a collection of the 4x rye-tiritcale hybrids and to determine their chromosome compositions. In the present study, karyotypes of the previously reported BC1F2s and BC1F3s were compared with that of the BC1F9 generation as obtained after several subsequent open pollinations. The genomic in situ hybridisation (GISH) allowed us to identify 133 introgression forms in which chromosome numbers ranged between 26 and 32. Using four DNA probes (5S rDNA, 25S rDNA, pSc119.2 and pAs1), the fluorescence in situ hybridisation (FISH) was carried out to facilitate an exact chromosome identification in the hybrid plants. The combination of the multi-colour GISH with the repetitive DNA FISH singled out five types of translocated chromosomes: 2A.2R, 4A.4R, 5A.5R, 5B.5R and 7A.7R among the examined BC1F9s. The reported translocation lines could serve as valuable sources of wheat chromatin suitable for further improvements of rye.

Different Chromosome Combinations on Tetra- and Hexaploid Level from Hybrids of Tetraploid Rye × Tetraploid Triticale

Different chromosome combinations at the tetra- and hexaploid level from the backcross hybrids 4x rye × 4x triticale with 4x triticale and 4x rye in the F2 generation were obtained. (1) Rye-cytoplasmic tetraploid triticale with 8 different chromosome constitutions in wheat genome. In the wheat genome, all chromosomes from A and B genomes, except the chromosome 3A, were represented. The ratio of wheat chromosomes in genomes A to B was from 1:6 (A1B6) to 4:3 (A4B3). (2) Lines of tetraploid rye with additional wheat chromosomes. Addition chromosomes in the lines of tetraploid rye were all chromosomes from the A genome, except chromosome 3A, and from B genome only the chromosomes 3B and 7B. The chromosome 3A occurred in a translocation form 3AS/5AL. (3) Lines of tetraploid rye with A/R and B/R substitutions. In the substitution lines occurred monosomics of chromosomes from A genome — 1A, 4A, 5A and 7A, and from B genome — 2B, 3B and 7B. Only two chromosomes from genome A — lA and 5A occurred in the disomic form. (4) Substitution-additive lines of tetraploid rye with wheat chromosomes. (5) Allohexaploids with ABRRRR genome constitution. A similar karyotype (one chromosome combination) — IB, 2A, 3B, 4B, 513, 6A and 7B — occurred in the wheat genome of all plants. (6) Tetraploid rye.

Molecular, physicochemical and rheological characteristics of introgressive Triticale/Triticum monococcum ssp. monococcum lines with wheat 1D/1A chromosome substitution.

Three sets of hexaploid introgressive triticale lines, with Triticum monococcum ssp. monococcum (cultivated einkorn wheat) genes and a bread wheat chromosome 1D substituted for chromosome 1A, and one set of secondary triticale lines were evaluated for grain and flour physicochemical and dough rheological characteristics in two generations (F7 and F8). Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) confirmed the 1D/1A chromosome substitution. The presence or absence of einkorn high-molecular-weight (HMW) glutenin subunits and the wheat Glu-D1d locus encoding the 5 + 10 subunits was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary zone electrophoresis, and allele-specific molecular markers. Significant differences were found among physicochemical properties (with the exception of the Hagberg falling number) of all introgressive Triticale/T. monococcum lines and the secondary triticale lines. The wheat 1D/1A chromosome substitution also affected these properties. The results showed that in all introgressive triticale lines, the protein and gluten content, Zeleny sedimentation value, and water absorption capacity, were increased. The rheological parameters estimated using micro-farinograph, reomixer, and Kieffer dough extensibility systems also showed an appreciable increase in dough-mixing properties, maximum resistance to extension (Rmax), and dough extensibility. Introgressive Triticale/T. monococcum lines with 5 + 10 subunits have particularly favorable rheological parameters. The results obtained in this study suggest that the cultivated einkorn genome Am, in the context of hexaploid secondary triticale lines and with a wheat 1D/1A substitution, has the potential to improve gluten polymer interactions and be a valuable genetic resource for triticale quality improvement.

Tolerance to Aluminium in Spring Triticales

The results from many experiments conducted over 4 years to determine the tolerance of 169 entries of Polish spring triticale and 63 entries of spring triticale from CIMMYT to aluminium are summarized. All experiments were conducted in nutrient solutions at varying aluminium concentrations (5, 15 and 25 ppm). The LD50 concentration of Al ions (ppm) was determined for each entry. All cultivars, strains and lines were classifed as very sensitive (LD50 25ppm).

An Attempt at Tetraploid Triticale Improvement

Since 1982 a small 4x triticale breeding programme has been carried out, aiming at creation of new lines with fertility, vigour and feeding value close to the 6x triticale cultivars and with the rye level of adaptation to marginal soil and weather conditions.