Using High-throughput Sequencing To Detect The Heteroplasmy Of COI Gene In Fig Wasps And Their Effects On Molecular Identification

Background: Heteroplasmy means the presence of more than one type of mitochondrial DNA(mt DNA) within an individual. To date, studies about heteroplasmy have focused on human health, ageing and tissue specificity. However, related researches in invertebrate are fewer, and the research hotspots are the status and origin of heteroplasmy. Results of previous research indicate that the genetic distance between heteroplasmic sequences can be up to 6%, which is much higher than the species identification level(2%) recommend by Hebert. However, there is no case study which concluded that heteroplasmy can mislead the DNA barcoding.Methods: In this study, we detect the heteropaslmy in different species, including those living in Ficus hispida: Ceratosolen solms(Agaonidae / Kradibiinae), Apocrypta bakeri(Pteromalidae / Sycoryctinae), Philotrypesis pilosa(Pteromalidae / Sycoryctinae), Philotrypesis sp., and those living in F. benjamina: Eupristina koningsbergeri(Agaonidae / Agaoninae), Philotrypesis sp.1, Philotrypesis sp.3, Philotrypesis sp.4, Philotrypesis sp.5, Walkerella benjamini(Pteromalidae / Otitesellinae), Walkerella sp.1, Sycophila sp.1(Eurytomidae / Eurytominae), Sycophila sp.2, Sycobia sp.2(Pteromalidae / Epichrysomallinae), Sycoscapter sp.1(Pteromalidae / Sycoryctinae), Acophila sp.1(Pteromalidae / Epichrysomallinae), Ormyrus sp.1(Ormyridae / Ormyrus).The next generation sequencing(NGS) provide life sciences with great help, and bring life sciences into the age of big data. In the field of heteroplasmy, the NGS have an advantage over the traditional methods: it can satisfy our demands for detecting the low frequence heteroplasmy. Our study was proceeded on the Roche 454 sequencing platform, and combine with mothur, Ultratranseq, Perl, CD-HIT, et al, we analyzed the heteroplasmy in fig wasps.Results:Based on bioinformatics analysis, the following main results are obtained:1) Mitochondrial heteroplasmy is widespread in fig wasp. We detect heteroplasmy in 92.3% of the tested individuals, covering 100% studied species.2) A low level mitochondrial heteroplasmy is detected in pollinating fig wasps, which cannot affect the accuracy of identification. Especially in Ceratosolen solmsi, only one of the four individuals shows mitochondrial heteroplasmy, and the rest individuals obtain no different sequences.3) In species with male polymorphism, the mitochondrial heteroplasmy is different by species. Species in the genus of Philotrypesis and Walkerella, we detect high level of mitochondrial heteroplasmy in two ways: 1, it keeps a greater K2 P genetic distance between the major and minor haplotypes(the COI fragments from one specimen may be clustered into different clades); 2, the proportion of minor haplotypes in higher(the proportion of minor haplotypes can be up to 71.8%). Especially in the case that different species share same haplotype, we must be careful when using COI as the tool of molecular identification. The proportion of minor haplotypes is lower in Sycobia sp.2, however, the K2 P genetic distance between the major and minor haplotypes is higher. Therefore, we cannot ignore the influence of minor haplotypes on molecular identification. In Sycophila sp.1 and Sycophila sp.2, the heteroplasmy is lower, and COI fragments can be used for molecular identification.4) In the species of Apocrypta bakeri, Acophila sp.1, Sycoscapter sp.1, and Ormyrus sp.1, the mitochondrial heteroplasmy is different by species, too. Sequences obtained from Acophila sp.1 are not enough for deep analysis. In Apocrypta bakeri, both of the proportion of minor haplotypes and K2 P genetic distance between the major and minor haplotypes is lower, and the heteroplasmy won’t have significant impact on molecular identification. The proportion of minor haplotypes in Sycoscapter sp.1 is higher. However, the K2 P genetic distance between the major and minor haplotypes is lower, which is cannot make a difference in molecular identification. In Ormyrus sp.1, both of the proportion of minor haplotypes and K2 P genetic distance between the major and minor haplotypes is higher, and we have to pay attention to using COI as the Barcoding marker.