A workflow of massive identification and application of intron markers using snakes as a model

Abstract

Relative to the commonly used mitochondrial and nuclear protein‐coding genes, the noncoding intron sequences are a promising source of informative markers that have the potential to resolve difficult phylogenetic nodes such as rapid radiations and recent divergences. Yet many issues exist in the use of intron markers, which prevent their extensive application as conventional markers. We used the diverse group of snakes as an example to try paving the way for massive identification and application of intron markers. We performed a series of bioinformatics screenings which identified appropriate introns between single‐copy and conserved exons from two snake genomes, adding particular constraints on sequence length variability and sequence variability. A total of 1,273 candidate intron loci were retrieved. Primers for nested polymerase chain reaction (PCR) were designed for over a hundred candidates and tested in 16 snake representatives. 96 intron markers were developed that could be amplified across a broad range of snake taxa with high PCR successful rates. The markers were then applied to 49 snake samples. The large number of amplicons was subjected to next‐generation sequencing (NGS). An analytic strategy was developed to accurately recover the amplicon sequences, and approximately, 76% of the marker sequences were recovered. The average p‐distances of the intron markers at interfamily, intergenus, interspecies, and intraspecies levels were .168, .052, .015, and .004, respectively, suggesting that they were useful to study snake relationships of different evolutionary depths. A snake phylogeny was constructed with the intron markers, which produced concordant results with robust support at both interfamily and intragenus levels. The intron markers provide a convenient way to explore the signals in the noncoding regions to address the controversies on the snake tree. Our improved strategy of genome screening is effective and can be applied to other animal groups. NGS coupled with appropriate sequence processing can greatly facilitate the extensive application of molecular markers.

Sequence capture across large phylogenetic scales by using pooled PCR‐generated baits: A case study of Lepidoptera

“…From this point of view, universal PCR‐based nuclear markers are becoming an essential resource for future phylogenetic studies rather than outdated tools. For those organism groups for which universal marker sets are already available, such as vertebrates (Shen, Liang, Feng, Chen, & Zhang, ), snakes (Li et al, ) and beetles (Che et al, ; Zhang et al, ), it will be easy to adopt our strategy to produce PCR baits for efficient sequence capture. However, universal marker sets are lacking for many large organism groups, such as Diptera, Hymenoptera and Orthoptera.…”

“…However, because PCR‐generated baits are normally long and can capture many flanking sequences, the final data length obtained from PCR baits is similar to that obtained from the AHE probes. In addition, bioinformatic screening of the genome data usually provides thousands of candidate loci for universal marker development for a certain organism group (Che et al, ; Li, He, Guo, Zhang, & Liang, ); thus, the number of applicable universal markers can easily be scaled up.…”

Integrative Methods Reveal Multiple Drivers of Diversification in Rice Paddy Snakes

“…We ampli ed cyt-b, ATPase, and PRLR using the polymerase chain reaction (PCR) protocols of Bernstein et al 2022 [27] . We followed nested PCR protocols [28,29] to amplify WFIKKN, VPS13B, AGBL5, BNC1, and RAG2. PCR products were visualized on a 1.5% agarose gel, and amplicons were cleaned with ExoSAP-IT (Applied BioSystems).…”

A sheep in wolf's clothing: Elaphe xiphodonta sp. nov. (Squamata, Colubridae) and its possible mimicry to Protobothrops jerdonii

“…Partial segments of the mitochondrial genes, 16S ribosomal RNA gene ( 16S ), cytochrome C oxidase 1 gene ( CO1 ) and Cytochrome b gene ( cytb ) were amplified. Nested PCR experiments were performed as described in Li et al (2017) . Primers used for PCR and sequencing followed Li et al (2020) , see Table 1 for details.…”