Analysis Of Mitochondrial Genome Sequences And Population Differentiation Of Several Invasion Leafminers

Liriomyza huidobrensis, L. sativae and L. trifolii are the three dangerous and most serious damage quarantine pest, which belong to Diptera, Agromyzidae. They had caused great losses and even to unproductive in agriculture production and export trade when they break out. The leafminers have the characters of small size, very similar morphology in closely related species, high degree of genetic differentiation even containing cryptic species, and it can also outbreak to be disaster in short time. Therefore, it is very important to study their genetic background and make clear their phylogenetic relationship for the quarantine and intergrated control. Meanwhile, as invasion pests, studies on their invasive pathway, geographic distribution and genetic diversity of knowledge, can provide proofs for more scientific basis for sustainable control. In this paper, the complete mitochondrial genomes of three leafminers were sequcened and phylogeny relationships of Diptera were reconstructed using the genebank data and ours. We also compared the geographic populations'differentiation in L. trifolii and L. huidobrensis. The main results were summarized as follows:1. Analysis of nuclear rDNA-ITS2 sequences, and mtDNA-COI, COII sequences of L. trifolii suggested that genetic differentiation appeared in different populations, The results showed that the different populations had separated into two clades: one was the United States clade, and the other was the Asia-Europe clade, while the low level differentiation were showed in domestic populations. According to our molecular data and the quarantine survey, for the first time, we deduced the invasion source and broadcast pathway of L. trifolii in our country, the populations of our country most probably were invaded from Europe.2. The results combined our study and Genbank data of mtDNA-COII showed different populations had genetic differentiation and can be divided into two clades, the USA population as one, and South America, Central America, Europe and Asia combined as the other. We also did more comprehensive analysis for the populations of our country and the result showed that there is still no genetic differentiation. Molecular data and Survey findings proved that the population of L. huidobrensis in China was invaded from Europe, which introduced from South America.3. Using the strategy of sub-PCR based on the long PCR, The complete mitochondrial genome of the three leafminers were sequenced and annotated. The genome lengths were 16141 bp, 16149 bp and 16238 bp respectively. The gene numbers and organization were similar to Drosphila yakuba contains one encoding region including 37 genes and one non-coding A+T-rich region, except that three additional tRNA genes (tRNAThr and tRNALeu(UUR), tRNAIle) were found in the A+T-rich region of L.trifolii and L. sativae respectively. The nucleotide compositions of the mitogenome of three leafminers were biased toward adenine and thymine, and the overall A+T contents were 78.06%, 78.1% and 78.23% respectively.4. For the first time, the quadruplet ATCA is used as protein start codon of mitogenome in insect, ND1 in L. trifolii and L. sativae which begins with GTG, the other protein initiation codons are ATN. Ten genes share the typical termination codons TAA and TAG, and ND4, CYTb of three species and ND5 in L. trifolii and L. sativae have incomplete codons of T. The usages of synonymous codons are showed a remarkable bias in the third codon position for A or T than to G or C. The amin acid content of Leu (15%-16%) is the highest and that of Cys (about 1%) is the lowest.5. All of tRNAs in three leafminers form the typical cloverleaf structure except for tRNASer (AGN), which has lost the DHU-arm. There are mismatchs with most of G-U in the secondary structures of majority tRNAs.6. The new additional tRNAs are predicted in A+T region of L.trifolii and L.sativae, the anticodons loop of tRNALeu(UUR) in L. trifolii contains a 50 bp intron located between the anticodon loop and arm. The tRNAThr in L. trifolii has one extra variable arm with a five-base-pair arm between the anticodon loop and the TψC loop. The additional tRNAIle containing one extra five-base-pairs variable arm is also found in the same region of L. sativae. The A+T region in both strands of three leafminers also contains a highly conserved polyT, which is correlated with the origin of mitochondrial genome replication.7. The overall phylogenetic relationships based on mitogenome of 31 Dipterans were reconstructed: the monophyly of Diptera, Brachyceran, Aristocera and Culicoideae are proved, which is consisitent with morphological classification. The relationships of Nematocera and interrelation of Brachycera were not fully resolved. Only in the BI tree, all the species of Nematocera priority clustered into one clade and this result strongly supports the monophyly of Nematocera.These results enrich the theory of phylogenetic relationship in three leafminers and Dipterans, make clear the broadcasting pathways, geodistribution and genetic diversity of L. trifolii and L. huidobrensis initially, and also provide the reliable proofs for building rapid molecular identification system. Our results will also play important role in quarantine diagnosis and sustainable control.