Aegilops crassa Boiss. repeatome characterized using low-coverage NGS as a source of new FISH markers: Application in phylogenetic studies of the Triticeae

Abstract

Aegilops tauschii Boiss. is polyploid grass species that grows in the eastern part of the Fertile Crescent, Afghanistan, and Middle Asia. It consists of tetraploid (4x) and hexaploid (6x) cytotypes (2n = 4x = 28, D1D (Abdolmalaki et al., 2019) XcrXcr and 2n = 6x = 42, D1D (Abdolmalaki et al., 2019) XcrXcrD2D (Adams and Wendel, 2005), respectively) that are similar morphologically. Although many Aegilops species were used in wheat breeding, the genetic potential of Ae. crassa has not yet been exploited due to its uncertain origin and significant genome modifications. Tetraploid Ae. crassa is thought to be the oldest polyploid Aegilops species, the subgenomes of which still retain some features of its ancient diploid progenitors. The D1 and D2 subgenomes of Ae. crassa were contributed by Aegilopstauschii (2n = 2x = 14, DD), while the Xcr subgenome donor is still unknown. Owing to its ancient origin, Ae. crassa can serve as model for studying genome evolution. Despite this, Ae. crassa is poorly studied genetically and no genome sequences were available for this species. We performed low-coverage genome sequencing of 4x and 6x cytotypes of Ae. crassa, and four Ae. tauschii accessions belonging to different subspecies; diploid wheatgrass Thinopyrum bessarabicum (Jb genome), which is phylogenetically close to D (sub)genome species, was taken as an outgroup. Subsequent data analysis using the pipeline RepeatExplorer2 allowed us to characterize the repeatomes of these species and identify several satellite sequences. Some of these sequences are novel, while others are found to be homologous to already known satellite sequences of Triticeae species. The copy number of satellite repeats in genomes of different species and their subgenome (D1 or Xcr) affinity in Ae. crassa were assessed by means of comparative bioinformatic analysis combined with quantitative PCR (qPCR). Fluorescence in situ hybridization (FISH) was performed to map newly identified satellite repeats on chromosomes of common wheat, Triticum aestivum, 4x and 6x Ae. crassa, Ae. tauschii, and Th. bessarabicum. The new FISH markers can be used in phylogenetic analyses of the Triticeae for chromosome identification and the assessment of their subgenome affinities and for evaluation of genome/chromosome constitution of wide hybrids or polyploid species.

qPCR as a Selective Tool for Cytogenetics

“…Repeat CL244 was identified in the genome of tetraploid Aegilops crassa (2 n = 28, DDXX) consisting of a D subgenome, similar to the D genome of diploid Aegilops tauschii (2 n = 14, DD) and an X subgenome of unknown origin, by analyzing the reads that did not align with the Ae. tauschii reference genome, i.e., by the application of a sequence filter [ 30 ]. Judging by the qPCR results, the copy number of CL244 in the genome of Ae.…”

“…The CL131 repeat was identified in the Ae. crassa genome during the search for X-subgenome-specific repeats (see paragraph CL244, [ 30 ]). This repeat is a satellite repeat, and judging by the bioinformatical analysis, it has a relatively moderate copy number.…”

“…In the study of the Ae. crassa subgenome markers [ 30 ], a low-copy tandem repeat CL239 that showed promise as an Ae. crassa -specific repeat was identified.…”

“…crassa and 1JS, 3JL, 5JS, 6JS, and 7JS in Th. bessarabicum [ 30 ]. Meanwhile, HRTR12 mainly hybridized to the sea buckthorn Y chromosome [ 33 ], while 17-202 was specifically localized to wheatgrass chromosomes, thus differentiating them from wheat chromosomes [ 28 ].…”

Intraspecific divergence of diploid grass Aegilops comosa is associated with structural chromosome changes

“…tauschii (2n=2x=14, DD), tetraploid and hexaploid Ae. crassa (2n=4x=28, D 1 D 1 X cr X cr and 2n=6x=42, D 1 D 1 X cr X cr D 2 D 2 (Kroupin et al 2022).…”