Identification of COS markers specific for Thinopyrum elongatum chromosomes preliminary revealed high level of macrosyntenic relationship between the wheat and Th. elongatum genomes

Abstract

Thinopyrum elongatum (Host) D.R. Dewey has served as an important gene source for wheat breeding improvement for many years. The exact characterization of its chromosomes is important for the detailed analysis of prebreeding materials produced with this species. The major aim of this study was to identify and characterize new molecular markers to be used for the rapid analysis of E genome chromatin in wheat background. Sixty of the 169 conserved orthologous set (COS) markers tested on diverse wheat-Th. elongatum disomic/ditelosomic addition lines were assigned to various Th. elongatum chromosomes and will be used for marker-assisted selection. The macrosyntenic relationship between the wheat and Th. elongatum genomes was investigated using EST sequences. Several rearrangements were revealed in homoeologous chromosome groups 2, 5, 6 and 7, while chromosomes 1 and 4 were conserved. Molecular cytogenetic and marker analysis showed the presence of rearranged chromosome involved in 6ES and 2EL arms in the 6E disomic addition line. The selected chromosome arm-specific COS markers will make it possible to identify gene introgressions in breeding programmes and will also be useful in the development of new chromosome-specific markers, evolutionary analysis and gene mapping.

Fig 1. Molecular cytogenetic analysis of the wheat-Th. elongatum addition lines.

Example of presence and identity verification of the wheat- Th. elongatum addition lines. A mitotic metaphase cell of the 2E disomic addition line after genomic in situ hybridization (GISH) (A) and fluorescence in situ hybridization (FISH) (B). In the wheat-Th. elongatum amphiploid (C and D), GISH (C) and FISH (D) detected the complete set of E-genome chromosomes, including chromosome 3E. In the GISH images the E genome was visualized in green, while in the FISH images the repetitive DNA probes pSc119.2, Afa family and pTa71 were visualized in green, red and yellow, respectively. Chromatin was nonspecifically stained with DAPI (blue). Scale bar = 10μm.

Fig 2. The comparison of the FISH karyotypes of Th. elongatum chromosomes 1E-7E identified in the genetic stocks used in this study.

Chromosomes 1E-7E (except 3E) from wheat-Th. elongatum disomic addition lines (A), E genome chromosome arms from wheat-Th. elongatum ditelosomic addition lines (B) and chromosomes 1E-7E (except 3E) from the wheat-Th. elongatum amphiploid line (C). Repetitive DNA probes pSc119.2, Afa family and pTa71 were visualized in green, red and yellow, respectively. The chromatin was nonspecifically stained with DAPI (blue). A horizontal white line indicates the position of the centromere. Scale bar = 10μm.

Fig 3. Fragment profiles of the COS markers on the CS- Th. elongatum addition lines.

Representative digital gel images for the assignment of markers to Thinopyrum elongatum chromosomes using the CS-Th. elongatum amphiploid (Amp), the parental wheat genotype Chinese Spring (CS) and the CS-Th. elongatum chromosome- (1E-7E) or chromosome arm (1–7 ES or EL) addition lines. (A) Polymorphic bands specific for one Th. elongatum chromosome as shown for marker TR335. (B) Polymorphic bands specific for several E-genome chromosomes shown for the marker c746642. (C) Different polymorphic bands specific for different E-genome chromosomes shown for the marker TR430. (D) Polymorphic bands assumed to be specific for 3E chromosome as shown for the marker TR62. Digital gel image were produced using PROSize 2.0 Software.

Fig 4. Wheat—Th. elongatum orthologous relationships.

The physical map of wheat pseudomolecules shows the positions of COS markers (on the left) on wheat chromosome groups 1–3, while the marker positions on Th. elongatum chromosomes are visualized by the coloured marker names (on the right). The marker positions on the wheat chromosomes were obtained by a BLASTn search for the source ESTs of the markers against the IWGSC wheat pseudomolecules (in bp values) and the start positions of the best hits were used for the map design. To indicate the chromosomal location in Th. elongatum the marker names were coloured according to the PCR results on wheat-Th. elongatum genetic stocks. Full colour shows markers located on the short arm, while half tone colour indicates location on the long arm. Bicolour markers indicate duplicated loci. In the wheat chromosomes arrowheads indicate the position of the centromere. *: The chromosomal location in Th. elongatum determined by the use of wheat-Th. elongatum amphiploid.

Fig 5. Wheat—Th. elongatum orthologous relationships.

The physical map of wheat pseudomolecules shows the positions of COS markers (on the left) on wheat chromosome groups 4–6, while the marker positions on Th. elongatum chromosomes are visualized by the coloured marker names (on the right). The marker positions on the wheat chromosomes were obtained by a BLASTn search for the source ESTs of the markers against the IWGSC wheat pseudomolecules (in bp values) and the start positions of the best hits were used for the map design. To indicate the chromosomal location in Th. elongatum the marker names were coloured according to the PCR results on wheat-Th. elongatum genetic stocks. Full colour shows markers located on the short arm, while half tone colour indicates location on the long arm. Bicolour markers indicate duplicated loci. In the wheat chromosomes arrowheads indicate the position of the centromere. *: The chromosomal location in Th. elongatum determined by the use of wheat-Th. elongatum amphiploid.

Fig 6. Wheat—Th. elongatum orthologous relationships.

The physical map of wheat pseudomolecules shows the positions of COS markers (on the left) on wheat chromosome group 7, while the marker positions on Th. elongatum chromosomes are visualized by the coloured marker names (on the right). The marker positions on the wheat chromosomes were obtained by a BLASTn search for the source ESTs of the markers against the IWGSC wheat pseudomolecules (in bp values) and the start positions of the best hits were used for the map design. To indicate the chromosomal location in Th. elongatum the marker names were coloured according to the PCR results on wheat-Th. elongatum genetic stocks. Full colour shows markers located on the short arm, while half tone colour indicates location on the long arm. Bicolour markers indicate duplicated loci. In the wheat chromosomes arrowheads indicate the position of the centromere. *: The chromosomal location in Th. elongatum determined by the use of wheat-Th. elongatum amphiploid.