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Possibility of numt co-amplification from gigantic genome of Orthoptera: testing efficiency of standard PCR protocol in producing orthologous COI sequences.

ABSTRACT: Mitochondrial DNA has been the preferential genome biodiversity studies. However, several factors contribute to its inadequacy. Numts constitute one of the main complications that prevent obtaining orthologous mitochondrial sequences. Orthoptera have been a model group in numt studies because of their huge genome size. In this study we aimed to; (i) test efficiency of standard PCR protocol in producing orthologous sequences of cytochrome C oxidase, (ii) study presence/absence of numts in several unstudied Orthoptera species, (iii) test if there is a threshold between the length of mtDNA targeted for amplification and possibility of encountering numts, and (iv) estimate reliability of the sequences in databases in light of these findings. For these aims we studied 38 species of Orthoptera representing different sublineages and genome sizes. DNA extracted from each sample was used to amplify five different fragments of COI region by standard PCR protocol. Sequenced PCR amplicons were checked for numt possibility by several different numt criteria. No sequences without numt signs were obtained for the first fragment. The number of samples with numt signs for the other four fragments differed between the suborders Ensifera and Caelifera. The percentage of samples with numt signs was higher in Caelifera than Ensifera for all fragments. The numt percentage considerably decreased for the longest two fragments. Numts are more prevalent in families with larger genome size. We arrived at the following conclusions: (i) numts are common in all members of Orthoptera, but, their prevalence differs among intra-lineages, especially more prevalent in Caelifera, (ii) there seems a correlation between numt rate and genome size, (iii) there is no threshold to avoid numt co-amplification, but, a 1,000 bp length may be a threshold for Ensifera, (iv) Folmer region of COI doesn't seem an appropriate marker for animal barcoding. Additionally, a phylogenetic tree produced from the numt sequences of fragment four detected in genus Anterastes suggested a paleonumt gained in generic ancestor a 3.5-4 times slower divergence rate for numt sequences.

SUBMITTER: Kaya S

PROVIDER: S-EPMC6260432 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Possibility of numt co-amplification from gigantic genome of Orthoptera: testing efficiency of standard PCR protocol in producing orthologous COI sequences.

Kaya Sarp S Çıplak Battal B

Heliyon 20181123 11

Mitochondrial DNA has been the preferential genome biodiversity studies. However, several factors contribute to its inadequacy. Numts constitute one of the main complications that prevent obtaining orthologous mitochondrial sequences. Orthoptera have been a model group in numt studies because of their huge genome size. In this study we aimed to; (i) test efficiency of standard PCR protocol in producing orthologous sequences of cytochrome C oxidase, (ii) study presence/absence of numts in several ...[more]

PMID: 30519651

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Project description:BackgroundDNA barcoding has been developed as a useful tool for species discrimination. Several sequence-based species delimitation methods, such as Barcode Index Number (BIN), REfined Single Linkage (RESL), Automatic Barcode Gap Discovery (ABGD), a Java program uses an explicit, determinate algorithm to define Molecular Operational Taxonomic Unit (jMOTU), Generalized Mixed Yule Coalescent (GMYC), and Bayesian implementation of the Poisson Tree Processes model (bPTP), were used. Our aim was to estimate Chinese katydid biodiversity using standard DNA barcode cytochrome c oxidase subunit I (COI-5P) sequences.ResultsDetection of a barcoding gap by similarity-based analyses and clustering-base analyses indicated that 131 identified morphological species (morphospecies) were assigned to 196 BINs and were divided into four categories: (i) MATCH (83/131 = 64.89%), morphospecies were a perfect match between morphospecies and BINs (including 61 concordant BINs and 22 singleton BINs); (ii) MERGE (14/131 = 10.69%), morphospecies shared its unique BIN with other species; (iii) SPLIT (33/131 = 25.19%, when 22 singleton species were excluded, it rose to 33/109 = 30.28%), morphospecies were placed in more than one BIN; (iv) MIXTURE (4/131 = 5.34%), morphospecies showed a more complex partition involving both a merge and a split. Neighbor-joining (NJ) analyses showed that nearly all BINs and most morphospecies formed monophyletic cluster with little variation. The molecular operational taxonomic units (MOTUs) were defined considering only the more inclusive clades found by at least four of seven species delimitation methods. Our results robustly supported 61 of 109 (55.96%) morphospecies represented by more than one specimen, 159 of 213 (74.65%) concordant BINs, and 3 of 8 (37.5%) discordant BINs.ConclusionsMolecular species delimitation analyses generated a larger number of MOTUs compared with morphospecies. If these MOTU splits are proven to be true, Chinese katydids probably contain a seemingly large proportion of cryptic/undescribed taxa. Future amplification of additional molecular markers, particularly from the nuclear DNA, may be especially useful for specimens that were identified here as problematic taxa.

Dataset Information

Possibility of numt co-amplification from gigantic genome of Orthoptera: testing efficiency of standard PCR protocol in producing orthologous COI sequences.

Publications

Possibility of numt co-amplification from gigantic genome of Orthoptera: testing efficiency of standard PCR protocol in producing orthologous COI sequences.

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