ANALYSIS OF KARYOTYPE STRUCTURE IN BRACHYPODIUM STACEI USING COMPARATIVE

CHROMOSOME PAINTINGHenry Shelonzek, Department of Plant Anatomy and Cytology,

University of Silesia in Katowice

Brachypodium distachyon (2n=10), a member of the Pooideae subfamily, is a small annual grass species native to southern Europe, northern Africa and southwestern Asia that has been established as a model for grasses to study various cellular and molecular biological processes. Due to its small genome (approx. 270 Mbp), close phylogenetic relationship with the Triticeae species, self-fertility, and a short lifecycle, B. distachyon has emerged as an excellent template to reference related cereals and grasses of economic importance. The bioinformatics analyses of genome sequencing data of several species allowed Salse et al. (2009) to infer that approximately 90 MYA existed an ancestral grass genome (n=5) from which today’s various monocot lineages have evolved. The ancestral genome underwent a whole genome duplication resulting in the formation of the paleo-tetraploid karyotype (n=10). The paleo-tetraploid was subsequently subjected to two chromosome fusions that led to the intermediate grass ancestor (n=12). From this intermediate genome evolved Ehrhartoideae (n=12), Poideae (n=7), and Panicoideae (n=10) 30-45 MYA. Of the 3 lineages, only Ehrhartoideae did not undergo further chromosome rearrangements. From that it can be deduced that among contemporary grass species, rice (an Ehrhartoideae representative) karyotype resembles the intermediate ancestral karyotype the most. The varying number of chromosomes in other major grass subfamilies also originated from an intermediate ancestral set of twelve chromosomes that had undergone chromosome fusions. According to the latest data from Salse et al. (2012), the 5 chromosomes of B. distachyon emerged through 7 centric fusion events. The study by IBI (2010) showed that B. distachyon has a high level of synteny to rice and sorghum, demonstrating that this species is a suitable template for grass functional genomic research.The genus Brachypodium comprises 14-19 species characterized by huge diversity in terms of basic chromosome number and ploidy level, as well as chromosome size and morphology. These features make it particularly well suited as a model to analyze the mechanisms of karyotype evolution in grasses.The aim of this study is to analyze the karyotype structure of B. stacei (2n=20) by comparative chromosome painting (CCP) using B. distachyon as a reference. This method utilizes hybridizing fluorescently labeled, chromosome specific probes of B. distachyon to the mitotic chromosomes of B. stacei. The painting probes were designed from rice syntentic chromosome regions described earlier by Febrer et al. (2010) & IBI (2010), for CCP of chromosome 1 (Bd1) and B. stacei. The CCP should enable direct visualization of B. stacei chromosomes homeologous to B. distachyon chromosome Bd1 and indicate the chromosomal rearrangements that discriminate the related genomes.The first step to initiate the study was to design the pools of B. distachyon BAC clones that would be used as probes and to check their specificity to particular regions of Bd1. This poster presents the resulats of these preliminary experiments.

Background

Seeds of B. distachyon and B. stacei were derived from the collections held by USDA-ARS and Aberystwyth University, UK, respectively. BAC clones derived from B. distachyon genomic DNA libraries (Febrer et al., 2010; IBI, 2010) were selected from the physical maps of Bd1 to be used in CCP of B. stacei. BAC DNA was extracted using standard alkaline lysis methods with QIAgen Plasmid Midi Kit (Roche, cat. no. 27106). Probes were labeled with digoxygenin-11-dUTP or tetramethyl-rhodamine-5-dUTP by nick translation. The protocol used for fluorescence in-situ hybridization (FISH) was adapted with minor modifications from Jenkins et al., (2007). Heterologous mapping was accomplished using a low stringency of 59%. Detection of the hybridization signals for digoxygenin-11-dUTP was done with anti-digoxigenin-fluorescein antibodies. In the case of tetramethyl-rhodamine-5-dUTP, the probe was directly visualized. The chromosomes were stained with DAPI (4’,6-diamidino-2-phenylindole). Images were visualized and captured using a CCD Retiga 2000e camera (QImaging) attached to an Olympus Provis AX epifluorescence microscope and were processed using Adobe Photoshop CC 2015.

Methods and Materials

• Obtain specific signals would entail mapping individual BAC clones to test which are showing unspecific signals and the removal of individual non-specific clones within each sub-pool.

• Further analysis of B. stacei karyotype with the BAC clones assigned to the remaining chromosomes of B. distachyon (Bd2-Bd5) to attain a better understanding of the chromosome rearrangements and karyotype evolution of the two species.

Future Perspectives

Results

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B. distachyon B. staceiChromosome painting of Bd1 was done by using BAC DNA probes designed to correspond to the rice homeologues of Bd1. The probes localized to chromosome loci specific for the probe however, unspecific hybridisation signals in the centromeres were also visible. This could be caused by too high amount of highly repeated DNA sequences within mapped BAC clones. Undesirable unspecific hybridization of the probes would not permit an accurate analysis of B. stacei karyotype, thus, increasing the specificity of the mapped clones is necessary.

Kariograms of B. distachyon and B. stacei Visual comparison of the karyotype morphology show B. distachyon’s metaphase chromosome being much bigger then B. stacei. Also, B. distachyon’s karyotype is more asymmetric. The chromosome size for B. distachyon ranges from 3.5 -7 µm, while in B. stacei the chromosomes vary from 1-3.5 µm.

B. distachyon chromosome 1 (Bd1) showing synteny to rice and the synteny breakpoints (sbp). The BAC DNA probes correspond to the rice chromosomes homeologous to the different segments of Bd1

Pool 3.1 - 3.3 | 12545551 bp | 27 clones | avg clone length 150648Pool 7.1 - 7,3 | 10857475 bp | 23 clones | avg clone length 145994Pool 6.1 - 6.6| 25468171 bp | 45 clones | avg clone length 146352 Pool 7.4 - 7.5 | 7363044 bp | 16 clones | avg clone length 141436Pool 3.4 - 3.7| 14983096 bp | 31 clones | avg clone length 156936

Bd1

Os3

3.1, 3.2, 3.3

Os7

7.1, 7.2, 7.3

Os6

6.1, 6.2, 6.3, 6.4, 6.5, 6.6

Os7

7.4, 7,5

Os3

3.4, 3.5, 3.6, 3.7

sbp1 sbp2 sbp3 sbp4

Short arm Long arm

Rice homeologues:

BAC pool IDs:

CEN

Scale bars: 5µm

Pool 3.1 – 3.7 probe labeled with tetramethyl-rhodamine-5-dUTP and mapped to Bd1. Scale bar: 5µm

B. distachyon B. distachyon

White arrows indicate unspecific hybridization signals located in the centromeres, possibly caused by hybridization of repeated DNA sequences.

Blue arrows indicate the hybridization signals specific to the main loci of probe 3

B. distachyon

• Febrer et al. (2010) PLOS ONE, 5(10): 13461• IBI (2010) Nature, 463: 763-768• Jenkins and Hasterok (2007) Nature Prot 2(1): 88-99• Salse et al. (2009) Oxford Journals Genome Biol Evol 4 (9): 917-928• Salse et al. (2012) Curr Opin Plant Biol, 15: 1–9

References