Formation and expression of pseudogenes on the B chromosome of rye

Abstract

B chromosomes (Bs) are dispensable components of the genomes of numerous species. In contrast with the prevalent view that Bs do not harbor genes, our recent sequence analysis revealed that Bs of rye (Secale cereale) are rich in gene-derived sequences. We compared these gene-like fragments of the rye B with their ancestral A-located counterparts and confirmed an A chromosomal origin and the pseudogenization of B-located gene-like fragments. About 15% of the pseudogene-like fragments on Bs are transcribed in a tissue-type and genotype-specific manner. In addition, B-located sequences can cause in trans down- or upregulation of A chromosome-encoded genic fragments. Phenotypes and effects associated with the presence of Bs might be explained by the activity of B-located pseudogenes. We propose a model for the evolution of B-located pseudogenes.

Figure 1.

Confirmation of B-Located Gene-Like Fragments. PCR performed using gDNA of rye and wheat with and without Bs and sorted rye B (A) and two wheat-rye B addition lines carrying either short- or long-arm iso-B chromosomes (B). Quality and equal quantity of DNA for different genotypes was shown by amplification using Bilby (a rye-specific sequence) and GAPDH (wheat and rye specific) primer pairs. Lengths of the expected amplicons are indicated.

Figure 2.

Chromosomal Locations of Gene-Like Fragment 12 by FISH. Mitotic metaphase (A) and meiotic pachytene (B) cells of rye with Bs after single-copy FISH with labeled extended gene-like fragment 12 (in green). FISH with the B-specific E3900 repeat (in blue) allowed the identification of Bs. Note multiple signals (arrow heads) of fragment 12 along the Bs in pachytene chromosome. A-localized FISH signals are indicated by arrows. The inset shows further enlarged B. DAPI, 4′,6-diamidino-2-phenylindole. Bar = 10 µm.

Figure 3.

Pseudogenization of B-Located Gene Fragments. (A) Schemata represent the sequence comparison between rye B-located pseudogene-like fragments (6 to 15) and their A-located parental counterparts. Percentage of similarity, transcribing region, and position of sequence polymorphisms are indicated. (B) Model for the evolution of B-located pseudogenes. The B of rye descended from As after a spontaneous whole or partial genome duplication. Meiotic recombination of proto-B with donor As became restricted. Proto- B still shows sequence similarity to the parental As. The increased gene dosage may affect gene expression and proto-B-located genes might have been suppressed by, for example, dosage compensation. Finally, B-located gene sequences became pseudogenized by mutations and accumulation of mobile and satellite DNA.

Figure 4.

Pseudogene-Like Fragments Are Conserved on the Bs from Different Rye Accessions. PCR performed with genomic DNA from rye (S. cereale subsp cereale from Turkey, Iran, China, Korea, and Japan, S. cereale subsp afghanicum from Afghanistan, and S. cereale subsp segetale from Pakistan) with and without Bs to determine the presence of the identified pseudogene-like fragments. B-specific repeat E3900 primers were used to demonstrate the presence of Bs. GAPDH-specific primers were used to show equal amounts of genomic DNA. Lengths of the expected amplicons are indicated.

Figure 5.

B-Located Pseudogene-Like Fragments Are Transcribed in a Tissue- and Species-Specific Manner. RT-PCR performed with cDNA derived from roots, leaves, and anthers of rye and of root and leaves of wheat with and without B chromosomes. Representative (sub)fragments are shown. PCR with genomic DNA was used as control. The presence of equal amounts of cDNA was tested by primers specific to GAPDH. Lengths of the expected amplicons from gDNA are indicated.

Figure 6.

Transcription Activity of B Pseudogene-Like Fragments across Different Rye Accessions. RT-PCR with cDNA of leaves from different rye accessions (S. cereale subsp cereale from Turkey, Iran, China, Korea, and Japan, S. cereale subsp afghanicum from Afghanistan, and S. cereale subsp segetale from Pakistan) with and without Bs. The transcription patterns were classified into four classes (I to IV). Class (I) (sub)fragments are expressed in +B plants only. Class (II) are not transcribed in 0B and +B plants. Class (III) are transcribed in all 0B and +B plants. Class (IV) shows complex expression patterns. Representative (sub)fragments for each group are shown. Lengths of the expected amplicons from gDNA are indicated.