Development of new microsatellite loci for wing dimorphic crickets Velarifictorus aspersus and their cross-utility in other Gryllidae species

Highlights

• Transcriptome sequencing was performed in wing dimorphic Velarifictorus aspersus.

• 7878 microsatellite loci were obtained from transcriptome data.

• 23 microsatellite markers were developed for V. aspersus.

• 3-22 microsatellite loci were successfully amplified in 6 other Gryllidae species.

Abstract

Microsatellite markers have been used extensively in studies of sperm competition and the genetic diversity of organisms. In the present study, we performed transcriptome sequencing for Velarifictorus aspersus and developed simple sequence repeat markers to identify valuable markers for genetic studies. The average total length of the V. aspersus transcriptome sequence was 7.70 Gb and it contained 7878 microsatellite loci. Sixty-four microsatellite loci were randomly selected for primer design and 39 loci were successfully amplified, among which 23 loci were polymorphic. The microsatellites were also tested in six other species from the Gryllidae family. Among the species belonging to the genus Velarifictorus, 22 loci were amplified in Velarifictorus micado, eight loci in Velarifictorus ornatus, and 3–6 loci in the other four cricket species.

Introduction

Microsatellite sequences, also known as simple sequence repeats (SSRs) or short tandem repeats, are repeating DNA sequences that are found widely in eukaryotic genomes (Akagi et al., 1996), where the basic repeating unit generally comprises 1–6 nucleotides (Tóth et al., 2000). SSRs are randomly distributed in the genome and their variations are caused by the occurrence of a base mismatch between the new strand and template strand in the microsatellite repeat region during DNA replication, which results in the insertion or deletion of one or several repeat units (Levinson & Gutman, 1987). Compared with other molecular markers, microsatellite markers have advantages such as good repeatability, rich polymorphism, convenient detection, co-dominant inheritance, and they are multi-allelic (Tuler et al., 2015, Powell et al., 1996, Clerc et al., 2005). Microsatellite markers has been used widely in the analysis of genetic diversity in species (Krishna et al., 2004), constructing genetic maps (Kirungu et al., 2018), quantitative trait loci mapping analysis (Cogan et al., 2005), paternity testing (Herlin et al., 2008), and other life science applications, such as detecting gene flow between populations (Rasero et al., 2010).

Crickets are good models for studying sexual selection and several researchers have identified paternity in the case of multiple copulations by using microsatellite markers (Bretman and Tregenza, 2005, Simmons and Beveridge, 2010). However, the universality of microsatellite markers is restricted by the genetic relationships among different groups. In some cricket species, nymphs rearing under different environmental conditions will emerge either as adults with long wings or adults with short wings. Long-winged (LW) adults have ability to fly, while short-winged (SW) adults are not able to fly. This phenomenon has been named as wing polymorphism, and is also common in other insect orders (Roff, 1986, Zera and Denno, 1997). There is a physiological trade-off between flight and reproduction in wing polymorphic insects according to the hypothesis proposed by Roff & Fairbairn (1991). Studies have shown that SW males have a greater advantage in competition for mating than LW males (Mitra et al., 2011, Zeng and Zhu, 2012), but the sperm competition capacity of wing polymorphic males has not been studied.

The cricket species Velarifictorus aspersus is widely distributed in China. This species exhibits distinct wing dimorphism and it is polygamous (Zeng et al., 2018, Zeng and Zhu, 2012). Therefore, it is an ideal material for studying sperm competition between wing dimorphic males. In a preliminary experiment, we performed PCR amplification for V. aspersus using 13 pairs of microsatellite primers from Teleogryllus oceanicus (Beveridge and Simmons, 2005, Simmons and Beveridge, 2010) and 16 pairs of microsatellite primers from Gryllus bimaculatus (Dawson et al., 2003), but none was successful. Thus, in the present study, we developed new polymorphic microsatellite loci for V. aspersus by using transcriptomic technology and tested their cross-utility in six other species from the Gryllidae family.