Mitochondrial genome recombination in somatic hybrids of Solanum commersonii and S. tuberosum | Scientific Reports – Nature.com

Posted: May 25, 2022 at 4:42 am

Complete mitochondrial genome assembly

The mitogenomes of St, Sc, and StSc were assembled into five to two subgenomes through de novo assembly using 5.3 to 6.6Gb PE reads. Each assembly was validated by conducting PCR analysis and sequencing (Tables S1 and S2, Fig. S1). The St mitogenome size was 756,058bp, and it was composed of five circular subgenomes of lengths 49,230 to 297,014bp. The total number of non-redundant genes was 78, consisting of 37 PCGs, 19 ORFs, 3 rRNAs, and 19 tRNAs (Table 1, Fig. S2A). The Sc mitogenome was 552,103bp in size with two subgenomes (338,427 and 213,676bp). The total number of non-redundant genes was 77, consisting of 37 PCGs, 20 ORFs, 3 rRNAs, and 17 tRNAs (Table 1, Fig. S2B). The StSc mitogenomes were 447,645bp in size with a major circular DNA of 398,439bp and a minor subgenome of 49,206bp. The total number of non-redundant genes was 77, consisting of 37 PCGs, 20 ORFs, 3 rRNAs, and 17 tRNAs (Table 1, Fig. S2C).

A total of 71 genes were shared among the three mitogenomes. Some genes were unique in each mitogenome: four ORFs (orf131, orf 190, orf 240, and orf 279), and three tRNAs (trnI-GAU, trnL-CAA, and trnV-GAC) were unique in the St mitogenome; five ORFs (orf109d, orf111, orf140, orf185, orf240) and one tRNA (trnfM-CAT) were unique in the Sc genome; and five ORFs (orf111, orf127, orf131, orf140, orf185) and one tRNA (trnV-GAC) were unique in the StSc mitogenome (Table 2).

Mitochondrial plastid DNA (MTPT) has been reported in various plants, such as Amborella trichopoda, Zea mays (maize), and Cynanchum wilfordii34,35,36. The degree of MTPT was examined by sequence comparison with the S. tuberosum plastome sequence (GenBank accession No. no. KM489056)37. Consequently, the St, Sc, and StSc mitogenomes were approximately 1.08.0%, 2.98.0%, and 3.14.0% considered as MTPT, respectively. Overall, approximately 1.08.0% were identified as MTPT (Table 1, Fig. S2).

Further, nuclear mitochondrial DNA (NUMT) has also been reported in various plants, such as Arabidopsis thaliana and Cucumis sativus (cucumber)38,39. NUMT was identified by sequence comparison with the S. tuberosum nuclear genome sequence (SolTub_3.0, https://www.ncbi.nlm.nih.gov/assembly/GCF_000226075.1/). Consequently, the St, Sc, and StSc mitogenomes were approximately 17.257.7%, 16.117.4%, and 10.116.3%, respectively, which were considered to be derived from or transferred to nuclear genomes accordingly. Overall, approximately 10.757.7% was identified as NUMTs. A total of 57.7% was identified in St subgenome 4, which has a very small genome size (Table 1, Fig. S2).

Homologous recombination (HR) can be mediated by repeat sequences in St, Sc, and StSc mitogenomes. The St, Sc, and StSc mitogenomes accounted for approximately 2.219.4%, 4.821.3%, and 5.725.9% of repeat sequences in which the repeat ratio was also positively correlated with the subgenome size (Table 1, Figs.1 and S2). The five St subgenomes exhibited diverse numbers of dispersed repeats: 300 (mitogenome coverage: 19.4%), 211 (15.2%), 41 (5.5%), 18 (2.2%), and 39 (4.9%) in each subgenome (Tables 1 and S5, Figs.1A and S2A). The two Sc subgenomes included 460 (25.9%) and 198 dispersed repeats (15.2%) (Tables 1 and S5, Figs.1B and S2B). Further, the two StSc subgenomes contained 480 (21.3%) and 39 (4.8%) dispersed repeats (Tables 1 and S5, Fig.1C and S2C). In contrast, tandem repeats were selected with adjacent sequences of at least two copies and up to 50bp. The St, Sc, and StSc mitogenomes had only 17, 20, and 16 tandem repeats, respectively (Table S6).

Chord diagram of three Solanum mitogenomes. (AC) represent the homologous regions of the subgenomes. R1 to R3 represent the large repeats that might cause homologous recombination among the corresponding subgenomes. St: S. tuberosum accession no. PT56, Sc: S. commersonii accession no. Lz3.2, StSc: somatic hybrid accession no. HA06-9.

Two large repeats (more than 1kb) were identified in the St subgenome 1. R1 was 11,916bp, and R2 was 7500bp. In contrast, St subgenome 2 had only R1, and subgenome 3 had only 1589bp of R3. Similarly, the R1 sequence co-existed in St subgenomes 1 and 2. The R2 repeat is shared between subgenomes 1 and 4 (Table S5, Figs.1 and S2), which might contribute to the HR between different subgenomes. The Sc mitogenomes had two multipartite structures, in which three large repeats of more than 1kb were identified (R1: 16,857bp, R2: 10,094bp, and R3: 1024bp), which might contribute to recombination events between subgenomes (Table S5, Figs.1 and S2). The StSc mitogenomes contain four large repeats (more than 1kb) (R1, 11,916bp; R2, 11,846bp; R3, 1643bp; and R4, 1024bp) that might contribute to subgenome reshuffling (Table S5, Figs.1 and S2).

We compared plastomes, mitogenomes, and nrDNAs among St, Sc, and StSc genomes. The StSc plastome was identical to Sc plastome37. Meanwhile, the StSc mitogenome shows a complicated structure with unique genes derived from both species (Table S3, Fig.2). Among 71 common genes, 21 PCGs (nad3, nad4, nad4L, nad5, nad6, sdh3, cox2, cox3, atp1, atp4, atp8, atp9, ccmB, rps3, rps4, rps12, rps13, rpl5, rpl10, rpl16, and mttB) were found identical across the three mitogenomes (denoted as green boxes on Fig.2) and their origin in the StSc genome could not be determined; 12 PCGs (nad1, nad2, nad7, nad9, sdh4, cob, cox1, ccmC, ccmFc, rps10, rpl2, and matR) were found identical with Sc (represented as sky-blue boxes in Fig.2) and 2 PCGs (atp6 and ccmFN) were identical with St (pink boxes in Fig.2). Therefore, it is likely that the majority of the somatic hybrid mitogenomes originated from Sc (Fig.2).

The origin of mitogenome recombination block in somatic hybrid (StSc) (A) Subgenome 1 of somatic hybrid mitogenome (B) Subgenome 2 of somatic hybrid mitogenome. The pink and sky-blue triangles on the black middle line indicate genes derived from S. tuberosum and S. commersonii, respectively. The green diamond boxes indicate genes of unknown origin.

GISH data using Sc genome probes revealed strong signals in 24 chromosomes but weak signals in the other 24 chromosomes in the StSc somatic hybrid (Fig.3A). We also assembled and compared 45S nrDNA cistron sequence of three species. For example, multiple aligned position at 191bp represents T genotype in St and C genotype in Sc. However, in StSc, it was identified that 75.6% of T and 24.4% of C were present. In conclusion, the overall 45S nrDNA sequences of StSc revealed both genotypes with average about 70 and 30 ratio for Sc and St, respectively (Fig.3B).

Detection of nuclear genome fusion in somatic hybrid. (A) GISH analysis of somatic hybrid (HA06-1 clone) using S. tuberosum specific-probes. The red signal of 24 arrows indicates the S. commersonii nuclear subgenomic distribution. (B) Schematic diagram of 45S ribosomal DNA cistron of Solanum species. StSc summary represents the percentage of St or Sc genotypes in the 45SnrDNA sequence.

In summary, St, a dihaploid of tetraploid cultivated potato, has five mitogenomes. Sc, a diploid wild potato, has two mitogenomes. Somatic hybrids developed via protoplast fusion of these two diploids contain the Sc-unique plastome37 but recombined mitogenomes and nuclear genomes derived from both St and Sc genomes (Fig.4).

Schematic diagram of mitogenome in parental species and their somatic hybrids. (A) S. tuberosum (St), (B) S. commersonii (Sc), and (C) somatic hybrid (StSc). S. tuberosum and S. commersonii have five and two subgenomes, respectively, which are fused into two subgenomes in the somatic hybrid generated by protoplast fusion. The origin of chloroplast genome in somatic hybrid has been determined based on sequence comparison among chloroplast genome sequences of parental species and that of the somatic hybrid.

A total of 35 PCGs were common across Solanaceae. The nonsynonymous substitution (Ka), synonymous substitution (Ks), and their ratios were calculated. The Ka values ranged from 0 to 0.119 with a 0.003 of median value. The nad4 and nad4L genes had the lowest Ka values, while atp6 had the highest Ka value. The Ks values ranged from 0.02 to 0.228 with a 0.01 of median value. Moreover, mttB and atp6 had the lowest and highest Ks values, respectively. Lastly, the Ka/Ks values ranged from 0 to 3.528 with a median value of 0.286 (Table S8, Fig.5A). A Ka/Ks value of more than 2 was observed due to the extremely low Ks value.

Mitochondrial gene diversity in Solanaceae family. (A) non-synonymous substitution (Ka) and synonymous substitution (Ks) values among the 12 Solanaceae species. Ka and Ks values were calculated with 35 protein-coding genes by CodeML program. (B) Variations of atp6 are shown by the phylogenetic tree and multiple comparisons of amino acid sequences. The conserved domain has been determined through NCBI BLASTP search.

Although the Ka and Ks values were generally low, ccmFc and mttB exhibited high Ka/Ks values of more than 1, indicating that these genes were positively selected during evolution (Fig.5A). Considering that atp6 showed a high mutation rate above 0.1. Ka and Ks values relative to the other genes, the amino-acid sequences corresponding to atp6 were compared among Solanaceae species, which revealed that amino acid sequences were variable at the N-terminus but conserved at the C-terminus (Fig.5B).

Phylogenetic trees were constructed using various programs, including RAxML, MEGA7, PhyML, and BEAST to examine the topology of the species. Trees treated with RAxML, PhyML, and BEAST displayed the same topology, while those treated with MEGA7 exhibited slightly different topologies (Fig. S3). In trees generated using RAxML representing an optimized topology (Figs.6 and S3), Solanaceae species were divided into two subfamilies, Solanoideae and Nicotianoideae, and the somatic hybrid exhibited a moderate branch between St and Sc. During the evolution of Solanaceae mitogenome, first, rps1 and rps19 were present in Solanaceae, however, these were omitted completely in Oleaceae. Next, rps7 was confirmed to be completely deleted in Solanaceae compared to Oleaceae. Lastly, ycf14 in all Nicotianoideae species was pseudogenized in the divergence period between Solanoideae and Nicotianoideae (Fig.6).

Phylogenetic relationship of 13 Solanaceae species using 35 protein-coding gene sequences commonly conserved in mitogenomes. The maximum likelihood tree was constructed using RAxML program with GTR++I model (based on jModelTest2) and a bootstrapping value of 1000. The bootstrap value (>=0.5) is shown on the node. Deleted genes and pseudogenes specifically within each group in the tree have been also shown by red and black boxes, respectively. Olea europaea in the Oleaceae family has been used as an out-group.

Here is the original post:
Mitochondrial genome recombination in somatic hybrids of Solanum commersonii and S. tuberosum | Scientific Reports - Nature.com

Related Posts