Sequencing And Analysis Of The Complete Mitochondrial Genomes Of Ceracris Nigricornis Nigricornis, Yunnanites Coriacea And Locusta Migratoria Migratoria

Abstract Mitochndrial genome is used as a useful model for structural, functional and evolutionary genomics studies, and has already made a great progress. There are 126 Arthropod species which the complete mitochondrial genome available in GenBank at present, but there are only two Orthoptera species, Locusta migratoria migratoioides and Gryllotalpa orientalis, which mitochondrial genomes have been sequenced. In order to enrich the Orthoptera mitochondrial genome data, and go further into structural and evolutionary studies of the mitochondrial genomes in Orthoptera insect, mitochondrial genomes of three Orthoptera species, Ceracris nigricornis nigricornis, Yunnanites coriacea and Locusta migratoria migratoria, belonging to three families, were sequenced, assemblyed, annotated and analyzed in details. The strategies of long-PCR and sub-PCR are used to finish the whole genome sequencing. After sequence assembly and annotation, three new mitochondrial genomes combined with the two other orthoptera species deposited in GenBank used to conduct a comprehensive comparative analysis, including the base composition and codon usage of mitochondrial genome protein-coding genes, secondary structure of tRNA and rRNA genes, conserved blocks and stem-loop structure of control regions, etc. The conclusions are mainly summarized as following.1. The size and A+T composition of mitochondrial genomes of C. nigricornis nigricornis, Y. coriacea, and L. migratoria migratoria are 15598bp, 75.23%, 15566bp, 74.60%, and 16053bp, 75.53% respectively. They all have a remarkably conserved set of 37 genes and a control region known as the (A+T)-riched region. The gene order and transcription direction are the same as that of L. migratoria migratoioides. The typical tRNA K-D order in Arthropoda has inverted to D-K order in these species. Compared with L. migratoria migratoioides and G. orientalis, the number and length of the integenic region and overlap region are much the same. The largest intergenic regions are all located between the tRNA^ser(s-UCN) and ND1.2. The mitochondrial protein-coding genes (PCGs) of C. nigricornis nigricornis, Y. coriacea, and L. migratoria migratoria show obvious bias. Compared the base compositon of the three codon positions of PCGs in five Orthoptera species, a common rule could be drawed as following. The second codon positions all have a higher T% than A%, and the T% is as much as twice of the A%. The third codon positions have the highest A+T composition, which is nearly 90%. The base composition of the third codon position between J-strand and N-strand varies a lot. J-strand is enriched with A and C, while N-strand is enriched with T and G. All the above has shown that the GC→AT evolutionary pressure is really strong, and J-strand and N-strand are under the different evolutionary pressure.3. The start codon usage of C. nigricornis nigricornis, Y. coriacea, and L. migratoria migratoria mitochondrial PCGs are much the same as that of L. migratoria migratoioides. I0 PCGs used the same ATN as the start codon, and only three genes have different start codons, COI, COIIand ND2. The start codons of COI gene in Y. coriacea, L. migratoria migratoria and L. migratoria migratoioides are special. They are CTG, ATTA and ATAA respectively. Majority of the 13 PCGs in these four species have a complete, ternimation codon, TAA or TAG, while several PCGs use an incomplete ternimation codon TA or T. However, in G. orientalis PCGs, ND2 has a GTG start codon, and there only 3 PCGs have complete ternimation codons.4. There are obvious biases in the both codon and amino acid usage in C. nigricornis nigricornis, Y. coriacea, L. migratoria migratoria, L. migratoria migratoioides and G. orientalis mitochondrial PCGs. NNU and NNA are the most frequently used codons. Leu, Ile, Ser and Phe have the highest composition of all the amino acids, and their total compositions in the five Orthoptera species are 43.8%, 43.91%, 43.07%, 43.00%, 42.69% respectively.5. All the 22 tRNA genes of C. nigricornis nigricornis, Y. coriacea, and L. migratoria migratoria mitochondrial genomes have a typical clver-leaf structure except tRNA^ser(AGN), which DHU arm could not form stable stem-loop structure. The length of the tRNA genes varies from 63bp to 72bp. Most of the mismatched base pair are G-U pairs.6. Compare the seconday structure of two rRNA genes in C. nigricornis nigricornis, Y. coriacea, and L. migratoria migratoria mitochondrial genomes, the structural differences are mainly located in the loop region, which indicates that the stem region is much more conserved than the loop region. 7. The conserved blocks in (A+T) riched region exist in all 3 species, including Block A, Block B, Block E1, Block E2 and Block F(except Y. coriacea). The sequence of the two blocks (E1 and E2) could form a stem-loop secondary structure, which the 5' flanking have a sequence consensus "...TTATAT", and the 3' flanking sequences share a sequence consensus "G(A)n(t)". The stem-loop secondary structure in C. nigricornis nigricornis, Y. coriacea are similar, but in the two locusta subspecies, the stem region is 3 base pairs longer. This stem-loop secondary structure may associate with the mtDNA replication origins.8. The L. migratoria migratoria mitochondrial (A+T) riched region is 1189bp in length. It contains four short nested highly conserved tandem repeated regions, repeat_region 1-1, repeat_region 1-2, repeat_region 2-1 and repeat_region 2-2, both two of which could form two longer repeats, repeat_regionl and repeat_region 2. There are 31 bp sequence spacer between the two longer repeats. The (A+T) riched region of L. migratoria migratoria stretches to the downstream of the 12S rRNA genes, which is the same as L. migratoria migratoioides.9. The general properties of the organization and structure characteristics of Orthoptera mitochondrial genome are drawed form the viewpoint of structural, comparative and evolutionary genomics through comparison of the three new genome data with the other two Orthoptera species data in GenBank, L. migratoria migratoioides and G. orientalis.This work is the first report about the complete mitochondrial genomes on C. nigricornis nigricornis, Y. coriacea, and L. migratoria migratoria. It adds three more Orthoptera complete mitochondrial genome sequences, and has accumulated useful information for mitochondrial phylogenomics research of insect.