Bioinfr: mtGenome

The complete mitochondrial genome of Taxus cuspidata (Taxaceae): eight proteincoding genes have transferred to the nuclear genome

© Kan et al. 2020

cite: Kan, Sheng Long , et al. “The complete mitochondrial genome of Taxus cuspidata (Taxaceae): eight protein-coding genes have transferred to the nuclear genome.” Bmc Evolutionary Biology 20.1(2020).
Paper: click here

© wikipedia

Abstract

BG: there have lots of Gymnosperms species but only a few of species have sequenced mitochondrial genome.
Result:

1. Complete mitogenome of Taxus cuspidate;
2. It lost many genes
3. Multiple tRNA and introns also have been lost
4. gene cluster could be less conserved
5. Few RNA sites were identified and may responsible for fewer introns and frequent gene losses

Background

1. Compared to chloroplast, only few of mitochondria genomes are available.
2. Previous study on mitogenome revealed that Cycans and Ginko are represent the ancestral type of seed plants.
3. Size; substitution; numerous of genes; introns and RNA editing sites; genome structure; repeats; turnover rates, and foreign sequence ratios are the arguments for mtGenome Comparing.
4. All other gymnosperms except Conifer II have mtGenome of represented species.
5. There are some ambiguity in relationship between Conifer, Pianceae, and Gnetales.
6. Our experiment and data are every valuable for the world.

Methods

Mitochodiral DNA isolation, total RNA extraction, sequencing and mitogenome assembly of Taxus cuspidata

graph TD;

st1(Yong leaves)
Ch1[DNase I]
op1(Density Gradient Centrifugation)

Ch2(RNAplant Plus Reagent)
st2(Yong seeds)
et(RNA)
lii(Illumina)
Oxf(Oxford Nanopore)

    st1-->op1;
    Ch1 -->|Remove DNA|op1;
    Ch2 --> st2;
    Ch1 -->|Remove DNA|st2;
    op1 -->et
    st2 -->et
    et -->|sequencing|lii
    et -->|sequencing|Oxf

Oxford Nanopore

1. First, approximately two micrograms of mitochondrial DNA was
   sheared by using Megaruptor
2. approximately one microgram of mitochondrial DNA was sonicated to ~ 500 bp using the Covaris M220 system

The sonicated DNA was purified using a TIANgel Midi Purification Kit, and a sequencing library was constructed using the NEBNext®
Ultra™ DNA Library Prep Kit for Illumina

Megaruptor: The Megaruptor ® was designed to provide researchers with a simple, automated, and reproducible device for the fragmentation of DNA from 2 kb - 75 kb.Shearing performance is independent of the source, concentration, temperature, or salt content of a DNA sample.

Illimina

graph TD;
R1((Raw Reads))
R2((H.Q. Reads))
Reads((Reads))
Con((Contigs))
Con2((Target Contigs))
Con3((Conf Contigs))
Con4((Contigs))
Con5((draft Contigs))
Con6((Contigs))
mt((mitogenome))
mt1((mitogenome))
mt2((mitogenome))
R1 --> |FastQC & Trimmomatic|R2
R2 -->|SPAdes|Con
Con-->|blast against mt protein|Con2
Con2 --> | BLASR to mtDB| Con3
Con3 --> |Canu v1.7.1|Con4
R2 -->|Canu v1.7.1|Con6
Con4 --> |Pilon v1.22|Con5
Reads-->|bowtie2|MUMmer
Reads-->|bowtie2|MUMmer2
Con4-->MUMmer
Con5-->MUMmer
Con5-->MUMmer2
Con6-->MUMmer2
MUMmer -->mt1
MUMmer2 -->mt2
mt1-->|MUMmer 3.0 and MAFFT v. 7|mt
mt2-->|MUMmer 3.0 and MAFFT v. 7|mt

mitogenome Annotation

protein and rRNA: blast+ against Cycas taitungensis, Ginkgo biloba
and Welwitschia mirabilis database.

• tRNA: tRNAscan-SE 2.0
• introns: RNAweasel tool
• ORFs: ORF Finder
• ORFs-> Blast2GO
• Visualization: OGDRAW

RNA editing site identification

• identify: RES-Scanner
• predicting: PREP-Mt

mt-genes to nuclear genome

• Depth of Sequencing Coverage: Bowtie2 v 2.2.9 & SAMtools v 1.6
• Realtime PCR: quantify the copy number of all protein coding genes
  (singlecopy nuclear gene LEAFY as an experimental control)

Identification of repeats, tandem repeats, Bpu elements and foreign sequences

• Repeasts: ROUSFinder.py
• tandem repeats: Tandem Repeats Finder
• Plastid-derived mtDNA (MTPTs) & Bpu-like elements: Guo et al. (span title here)
  • MIPTs: blastn (against to genome and mitogenome)
  • Bpu-like elements: blastn (using the Cycas Bpu consensus sequence as a query)
• nuclear-derived repetitive sequences: RepeatMasker web server

Shared DNAs and gene cluster analyses

(mtDNA shared between species)
blastn with a word size of 7 and an e-value cutoff of 1 × 10− 6.
Syntenic relationships were generated using Circos v. 0.69

Evolutionary rate heterogeneity test

1. mitochondrial protein-coding genes
2. Ran et al. :

• Sequence alignment;
• unreliable sequence alignment filtering;
• synonymous (d_S);
• nonsynonymous length calculations (d_N);
• absolute nonsynonymous(R_N);
• synonymous rate (R_S).

3. Effects of C-to-U RNA editing:

• predicted editing sites were excluded

Result

Mitogenome size and gene and intron contents of Taxus cuspidata

Category	Size
Size	468,924bp
gene	46
protein	32
tRNA	10
rRNA	3
small subunit ribosomal protein	4
large subunit ribosomal protein	2
cytochrome C biogenesis	4
transport membrane protein	1
maturaserelated protein	1
Intron
group I introns	0
group II introns	15 (cox2, nad1,nad2, nad4, nad5 and nad7 )
cis-spliced	4
trans-spliced	11
tRNA
native tRNA	9
plastids tRNA	1
rRNA	copies
rrn5 & rrn26	1
rrn18	2

Category	Length
genes	225 -4104bp
exons	22 - 1224bp
introns	804 - 2461bp

nine, one, one, three, and five encoding mitochondrial respiratory chain complexes I, II, III, IV, and V, respectively.

Variation in gene and intron

Comparing groups: Taxus, Pinus, Welwitschia, Cycas and Ginkgo.

	Cycas	Ginkgo	Pinus	Welwitschia	Taxus
mitogenome Size	414,903	346,544	1,191,054	978,846	468,924
GC%	46.9	50.4	47	53	50.39
tRNAs	27	23	12	8	10
rRNAs	3	3	6	3	4
Protein coding	41	41	41	29	32
ORF	3945 (414,858 bp)	3944 (323,967 bp)	10,587 (1,191,015 bp)	11,171 (978,799 bp)	3923 (468,857 bp)
Introns	26	25	26	10	15
Predicted edit sites	1206	1306	1179	225	1102
Repeats (kb)	80 (19.2%)	32 (9.3%)	170 (14.2%)	50 (5.0%)	62 (13.2%)
Tandem repeats (kb)	22 (5.3%)	3.6 (1.1%)	71 (6.0%)	24 (2.5%)	48 (10.2%)
Plastid-derived (kb)	19 (4.6%)	0.3 (0.1%)	5.6 (0.5%)	7.9 (0.8%)	0 (0%)
Nuclear-derived repetitive (kb)	3.4 (0.8%)	1.9 (0.6%)	5.3 (0.5%)	2.5 (0.3%)	3.5 (0.8%)
Lost Genes				8+1	8+2

Homologous transcripts of eight of the nine lost mitochondrial genes (excluding rpl10) were found in the transcriptome of Taxus cuspidata


© Kan et al. 2020

RNA editing site abundance and efficiency

RES-Scanner

• efficiency:
  • 0.05; 974 C-to-U
  • 791 them are on protein-coding genes
    • 730 were in coding regions
    • 61 were in introns
  • 1 in tRNA
  • 2 in rRNA
  • 180 in intergenic regions
• first and second codon positions have higher editing efficiencies
• nonsilent editing sites have higher editing efficiencies than silent sites

PREP-Mt: only nonsilent RNA editing sites in proteincoding genes could be predicted

• cutoff score: 0.2
• 1102 C-to-U editing sites
• only 474 were identical
• …

© Kan et al. 2020

Structural and gene cluster dynamics

1. DNA shared by Cycas and Ginkgo up to 200 bp (about 1/2)
2. Shared by Cycas and other three are short. (30 ~ 50)

Repeats, tandem repeats, and foreign DNA sequences

1. Cycas and Pinus mitogenomes contain more dispersed repeats
2. In addition, most repeats had more than two copies in the Cycas and Pinus
3. Plastid-derived sequence (> 100bp) not found in Taxus.
4. nuclear-derived repetitive sequences found in five species.
5. …

Variation in nucleotide substitution rates

although eight putatively transferred genes in Taxus and Welwitschia were still found in the mitogenomes of Cycas, Ginkgo and Pinus, their synonymous and nonsynonymous substitution rates were higher than those of other mitochondrial genes

Discussion

Separate losses of multiple mitochondrial protein-coding genes in Taxus and Welwitschia

• Only two genes were lost in the first approximately 300 myr of land plant evolution if maturase is not considered
• parallel gene losses documented in hornworts, lycophytes, and ferns also happened in more recent times
• In angiosperms, a large number of protein-coding genes have been lost in some lineages,
• …

Separate losses of multiple mitochondrial tRNA genes in Pinaceae, Taxus and Welwitschia

• all gymnosperms have three rRNA genes in their mitogenomes.
• the number of tRNAs differs greatly

Frequent losses and cis- to trans-splicing of introns in the mitogenomes of gymnosperms

Both the ancestral angiosperm and gymnosperm mitogenomes contain 26 group II introns.
Cycas and Ginkgo, have 26 and 25 introns, whereas only ten introns are found in Welwitschia.

Mechanisms for intron loss:

• genomic deletion
• exonization
• gene conversion
• EGT: rpl2i846 and rps10i235
• retroprocessing (a reverse transcriptase-mediated model)

Gene clusters could be less conserved in gymnosperms, and transposable elements and specific repeats are rare in the Taxus and Welwitschia mitogenomes

…

The number of RNA editing sites is not correlated with the GC content of mitochondrial genes

Taxus cuspidata: results showed that all editing sites are C-to-U conversions
…

Size variation in gymnosperm mitogenomes is still a mystery

…