Isolation and characterization of chromosomal markers in Poa pratensis

Abstract

Background Poa pratensis L. is a turf grass and forage crop used worldwide. Being a facultative apomictic species, P. pratensis has a highly variable chromosome number. Chromosomal markers constitute a powerful tool for chromosome identification and for various aspects of genomic research. However, currently, no chromosomal markers are available for P. pratensis.ResultsFour novel chromosome markers were isolated from a screen of Cot-1 DNA libraries, combined with fluorescence in situ hybridization (FISH) in Poa pratensis. Three tandemly repetitive sequences (PpTR-1, PpTR-2, and PpTR-3) were characterized as subtelomeric. Monomers of 318 bp, 189 bp and 189 bp were identified in PpTR-1, PpTR-2, and PpTR-3, respectively. One tandemly repetitive sequence (PpCR-1) was shown to be centromeric or pericentromeric, and it had a monomer of 27 bp. The distribution patterns of PpTR-1, PpTR-2, and PpTR-3 were highly conserved across different P. pratensis cultivars and in the distantly related Poa species, whereas PpCR-1 was conserved across different P. pratensis cultivars, but less conserved across Poa species.ConclusionIn this study, we report the identification and characterization of four novel chromosomal markers in P. pratensis. These chromosomal markers are powerful tools for accurate assessment of chromosome count, genomic and phylogenetic analyses, as well as studies of apomixis in P. pratensis. Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-017-0307-7) contains supplementary material, which is available to authorized users.

Identification and characterization of satellite DNAs in Poa L.

“…Examination of chromosomal distribution of the four above satellites carried out in the diploid form of P. malaca with the chromosome number of 14 . Since the satellite 365 bp and 189 bp showed the high similarity with the previous identified tandem repeats Pp Tr-1, Pp Tr-2 and Pp Tr-3 in P. pratensis , the clone sequence probes Pp Tr-1 and Pp Tr-3 [ 18 ] were used for representing the satellite 365 bp and 189 respectively in this study. Oligonucleotide FISH probes were designed for Poa -362 and Poa -353 from the sequences of the monomers using DNAman software package (Lynnon Biosoft, Quebec, Canada) (Table 1 ).…”

“…Metaphase chromosomes of root tip cells were used for FISH analysis. A detailed chromosome preparation was according to Zhao et al [ 18 ].…”

“…The 5S rDNA was amplified by PCR using genomic DNA of P. pratensis according to Fukui et al [ 24 ]. The satellite DNA of Pp Tr-1 and Pp Tr-3 were amplified from the previous made clones [ 18 ]. The 5S rDNA, Pp Tr-1 and Pp Tr-3 were by a random primer labelling method, described by Dou et al [ 25 ].…”

“…Two other oligonucleotide designed to represent the newly identified satellite DNAs were end-labelled with FAM (green) or TAMRA (red). The FISH hybridization procedure and micrometry followed Zhao et al with minor modification [ 18 ]. Before hybridization reaction, the hybridization mixture with oligonucleotide probes was placed directly onto the denatured slide preparation, rather than the hybridization mixture with the ddsDNA probe was denatured in boiling water for 5 min.…”

“…Four satellite DNAs were isolated from P. pratensis by construction and screening cot-1 libraries, and the conservation and variability of these satellites was tested in only one more related Poa species [ 18 ]. It is still unknown the phylogenetic distribution of these satellite DNAs in other more Poa species.…”