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Exploiting repetitive sequences and BAC clones in Festuca pratensis karyotyping.

ABSTRACT: The Festuca genus is thought to be the most numerous genus of the Poaceae family. One of the most agronomically important forage grasses, Festuca pratensis Huds. is treated as a model plant to study the molecular mechanisms associated with tolerance to winter stresses, including frost. However, the precise mapping of the genes governing stress tolerance in this species is difficult as its karyotype remains unrecognized. Only two F. pratensis chromosomes with 35S and 5S rDNA sequences can be easily identified, but its remaining chromosomes have not been distinguished to date. Here, two libraries derived from F. pratensis nuclear DNA with various contents of repetitive DNA sequences were used as sources of molecular probes for fluorescent in situ hybridisation (FISH), a BAC library and a library representing sequences most frequently present in the F. pratensis genome. Using FISH, six groups of DNA sequences were revealed in chromosomes on the basis of their signal position, including dispersed-like sequences, chromosome painting-like sequences, centromeric-like sequences, knob-like sequences, a group without hybridization signals, and single locus-like sequences. The last group was exploited to develop cytogenetic maps of diploid and tetraploid F. pratensis, which are presented here for the first time and provide a remarkable progress in karyotype characterization.

SUBMITTER: Majka J

PROVIDER: S-EPMC5462415 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Exploiting repetitive sequences and BAC clones in Festuca pratensis karyotyping.

Majka Joanna J Książczyk Tomasz T Kiełbowicz-Matuk Agnieszka A Kopecký David D Kosmala Arkadiusz A

PloS one 20170607 6

The Festuca genus is thought to be the most numerous genus of the Poaceae family. One of the most agronomically important forage grasses, Festuca pratensis Huds. is treated as a model plant to study the molecular mechanisms associated with tolerance to winter stresses, including frost. However, the precise mapping of the genes governing stress tolerance in this species is difficult as its karyotype remains unrecognized. Only two F. pratensis chromosomes with 35S and 5S rDNA sequences can be easi ...[more]

PMID: 28591168

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Project description:Radish (Raphanus sativus L., 2n?=?2x?=?18) is a major root vegetable crop especially in eastern Asia. Radish root contains various nutritions which play an important role in strengthening immunity. Repetitive elements are primary components of the genomic sequence and the most important factors in genome size variations in higher eukaryotes. To date, studies about repetitive elements of radish are still limited. To better understand genome structure of radish, we undertook a study to evaluate the proportion of repetitive elements and their distribution in radish.We conducted genome-wide characterization of repetitive elements in radish with low coverage genome sequencing followed by similarity-based cluster analysis. Results showed that about 31% of the genome was composed of repetitive sequences. Satellite repeats were the most dominating elements of the genome. The distribution pattern of three satellite repeat sequences (CL1, CL25, and CL43) on radish chromosomes was characterized using fluorescence in situ hybridization (FISH). CL1 was predominantly located at the centromeric region of all chromosomes, CL25 located at the subtelomeric region, and CL43 was a telomeric satellite. FISH signals of two satellite repeats, CL1 and CL25, together with 5S rDNA and 45S rDNA, provide useful cytogenetic markers to identify each individual somatic metaphase chromosome. The centromere-specific histone H3 (CENH3) has been used as a marker to identify centromere DNA sequences. One putative CENH3 (RsCENH3) was characterized and cloned from radish. Its deduced amino acid sequence shares high similarities to those of the CENH3s in Brassica species. An antibody against B. rapa CENH3, specifically stained radish centromeres. Immunostaining and chromatin immunoprecipitation (ChIP) tests with anti-BrCENH3 antibody demonstrated that both the centromere-specific retrotransposon (CR-Radish) and satellite repeat (CL1) are directly associated with RsCENH3 in radish.Proportions of repetitive elements in radish were estimated and satellite repeats were the most dominating elements. Fine karyotyping analysis was established which allow us to easily identify each individual somatic metaphase chromosome. Immunofluorescence- and ChIP-based assays demonstrated the functional significance of satellite and centromere-specific retrotransposon at centromeres. Our study provides a valuable basis for future genomic studies in radish.

Project description:BackgroundSpecies of the Festuca and Lolium genera, as well as intergeneric Festuca × Lolium (Festulolium) hybrids, are valuable fodder and turf grasses for agricultural and amenity purposes worldwide. Festulolium hybrids can merge in their genomes agronomically important characteristics. However, in polyploid plants, especially in allopolyploids, the hybridization of divergent genomes could contribute to various abnormalities, such as variability in chromosome number, structural rearrangements, and/or disorders in inheritance patterns. Here we studied these issues in allotetraploid Festuca pratensis × Lolium perenne hybrids.ResultsCytogenetic procedures, including fluorescent in situ hybridization, genomic in situ hybridization, and molecular markers - inter-simple sequence repeats (ISSR) were exploited. This cytogenetic approach indicated the dynamics in the number and distribution of ribosomal RNA genes and structural rearrangements for both parental genomes (Festuca and Lolium) in hybrid karyotypes. The separate analysis of F. pratensis and L. perenne chromosomes in hybrid plants (F2-F3 generations of F. pratensis × L. perenne) revealed the asymmetrical level of rearrangements. Recognized structural changes were mainly located in the distal part of chromosome arms, and in chromosomes bearing ribosomal DNA, they were more frequently mapped in arms without this sequence. Based on the ISSR markers distribution, we found that the tetrasomic type of inheritance was characteristic for the majority of ISSR loci, but the disomic type was also observed. Nonetheless, no preference in the transmission of either Festuca or Lolium alleles to the following generations of allotetraploid F. pratensis × L. perenne hybrid was observed.ConclusionOur study reports cytogenetic and molecular genotyping of the F. pratensis × L. perenne hybrid and its following F2-F3 progenies. The analysis of 137 allotetraploid F. pratensis × L. perenne hybrids revealed the higher level of recombination in chromosomes derived from F. pratensis genome. The results of ISSR markers indicated a mixed model of inheritance, which may be characteristic for these hybrids.

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Exploiting repetitive sequences and BAC clones in Festuca pratensis karyotyping.

Publications

Exploiting repetitive sequences and BAC clones in Festuca pratensis karyotyping.

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