Multicopy genes uniquely amplified in the Y chromosome-specific repeats of the liverwort Marchantia polymorpha

Abstract

Sex of the liverwort Marchantia polymorpha is determined by the sex chromosomes Y and X, in male and female plant, respectively. Approximately half of the Y chromosome is made up of unique repeat sequences. Here, we report that part of the Y chromosome, represented by a 90-kb insert of a genomic clone pMM2D3, contains five putative genes in addition to the ORF162 gene, which is present also within the Y chromosome-specific repeat region. One of the five putative genes shows similarity to a male gamete-specific protein of lily and is expressed predominantly in male sex organs, suggesting that this gene has a male reproductive function. Furthermore, Southern blot analysis revealed that these five putative genes are amplified on the Y chromosome, but they also probably have homologs on the X chromosome and/or autosomes. These observations suggest that the Y chromosome evolved by co-amplifying protein-coding genes with unique repeat sequences.

Figure 1

Schematic diagram of the structure of pMM2D3. The insert of the plasmid is displayed with its SP6-end on the left. Alignments of sequence contigs are shown by the lower horizontal bars labeled as RS, RT, R1, R2, R3 and R4. Sequences of R1, R2, R3 and R4 consisted of the Y chromosome-specific repeat sequences. Approximate sizes of contigs are given in parentheses in kb. The order of contigs in brackets was not determined. Putative genes are indicated by closed boxes above (left-to-right orientation) and below (right-to-left orientation) the line. Recognition sites for PacI (P) and SfiI (S) determined by restriction digestion and electrophoresis are indicated by vertical lines. Clusters of the Y chromosome- specific repeat sequences are indicated by open boxes. The exact position of M2D3.5 within the clone is not known because of the repeat sequences, but its orientation is predicted from the orientation of the repeat sequences at the proximal ends of RS and RT.

Figure 2

Structures of the 2.0-kb and 1.8-kb BamHI repeat units newly identified in pMM2D3, in comparison with the 2.4-kb and 1.5-kb BamHI repeat units of pMM4G7. As in the 2.4-kb and 1.5-kb BamHI repeat units, the 2.0-kb and 1.8-kb BamHI repeat units also consist of common subrepeats, MboI (red) and HaeIII elements (blue). Other sequences are color-coded (white, yellow and green) according to their respective similarities.

Figure 3

Multiple amino acid sequence alignment of the pyridoxal phosphate binding site in M2D3.1 and its related alliin lyase-like proteins. The numbers in parentheses indicate the positions in the respective sequence. Amino acid residues identical to those of M2D3.1 are indicated by colons. The lysine residue suggested to be the pyridoxal phosphate binding site is indicated by an arrow. AtAlh, alliin lyase homolog of A.thaliana (GenBank accession number T05567); AcAl, Allium cepa alliin lyase (AAA32639); AtuAl, A.tuberosum alliin lyase (BAA20358).

Figure 4

Alignment of B3 DNA-binding domains found in M2D3.2. (A) Schematic illustration of M2D3.2 with two B3 DNA-binding domains, B3-A and B3-B. (B) Amino acid sequence alignment of B3 DNA-binding domains found in M2D3.2 and other proteins. The numbers in parentheses indicate the positions in the respective sequences. Amino acid residues identical to those of B3-A, are indicated by colons. Gaps are indicated by dashes. pB3, putative DNA binding protein of A.thaliana (GenBank accession no. AAC34233); RAV1, related to ABI3/VP1 1 DNA-binding protein of A.thaliana (BAA34250); RAV2, related to ABI3/VP1 2 DNA-binding protein of A.thaliana (BAA34251); ARF9, auxin response factor 9 of A.thaliana (AAD24427); VP1, VIVIPAROUS1 protein of maize (P26307).

Figure 5

Amino acid sequence alignment of the PAH domains found in M2D3.6 and other proteins. The numbers in parentheses indicate the positions in the respective sequence. Amino acid residues identical to those of the PAH domain of M2D3.6 are indicated by colons. Gaps are indicated by dashes. OsUP, unknown protein of rice (GenBank accession no. AAG03087); AtTRl, transcription regulator-like protein of A.thaliana (T51447); MmSin3A, Sin3 transcription regulator homologous protein of Mus musculus (AAA89119); ScSin3, Sin3 transcription regulator protein of Saccharomyces cerevisiae (RGBYS3).

Figure 6

The sex specificity and copy number of the five putative genes. (A) Sex specificity of the five putative genes was examined by genomic PCR with primer pairs designed for the conserved regions of the respective five genes. CDPK served as a quality control for the genomic DNAs. (B) The copy numbers of the five putative genes were examined by genomic Southern blot analyses. Genomic DNAs of male (M) and female (F) plants were digested with BamHI, and were probed with DNA fragments of the respective ORFs and CDPK. Closed circles indicate intense signals detected specifically in male DNAs. Open circles indicate signals detected in both male and female DNAs. Sizes of signals are given in kb on the right.

Figure 7

RT–PCR analyses of the putative genes. Poly(A)⁺ RNAs isolated from male thalli (lanes MT), male sex organs (lanes MS), female thalli (lanes FT) and female sex organs (lanes FS) were reverse-transcribed, and the resulting cDNAs were used as templates (lanes +). The same amounts of poly(A)⁺ RNA but without the RT reactions were used for control (lanes –). Positive control PCRs were performed using 1 ng of pMM2D3 plasmid DNA as a template (P). Negative control PCRs were carried out without added DNA (N). Sizes of PCR products are given in bp on the right. Primer pairs for RT–PCR here were the same as those used for genomic PCRs.

Figure 8

Northern blot analysis of M2D3.4. (A) Five micrograms of poly(A)⁺ RNA from male thalli (lane MT), male sex organs (lane MS), female thalli (lane FT) and female sex organ (lane FS) were blotted and probed with a ³²P-labeled DNA covering M2D3.4. (B) The same membrane was reprobed with the CDPK gene, which is constitutively expressed in male and female. Sizes of signals are given in kb on the right.