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Budgerigar mirnas analysis

Abstract

The budgerigar (Melopsittacus undulatus) may be the most generally studied parrot species which provides as an excellent model of tendencies and neuroscience. Many sex differences in tissue morphogenesis and behavior derive from differential gene expression. MicroRNAs (miRNAs) are endogenous brief non-coding RNA molecules that may regulate the post-transcriptionally gene expression. Commonly, miRNAs have pivotal roles in gonadal differentiation and production in animals. Even so, biological capabilities of gonadal miRNAs in birds continue to be woefully understudied. Studies on sex-biased expression of miRNAs in avian gonads are limited, and little is well known about M. undulates. In today’s study, we sequenced two small non-coding miRNAs libraries created from the gonads of adult male and female budgerigars employing illumina paired-end sequencing technology, respectively. The expression profiles of miRNAs from ovary and testis were built and a phylogenetic analysis was performed. We received 211 known and 41 novel miRNAs, and randomly validated five miRNAs. In addition, a total of 17 drastically differentially expressed miRNAs were identified. Moreover, 60 gender-specific miRNA that may come right down to sexual differentiation were detected in the gonads that have been subjected to functional analysis with Head out annotation and KEGG pathway evaluation. Notably, this study also discovered that the miR-215 precursor sequences could provide you with some phylogenetic information, specifically supporting the close relationships among three avian species. In conclusion, our work for the first time provided numerous features of sex-biased miRNAs of the budgerigar, enriched the database in avian miRNAome and facilitated further functional research.

Keywords: MiRNA, sex-biased, gonads, adult, Melopsittacus undulates.

  1. Introduction

MiRNAs are tiny non-coding RNAs playing essential parts in regulating post-transcriptionally in miscellaneous sorts of animals and vegetation (Bartel, 2004, 2009; Chen and Rajewsky, 2007). There are almost thirty thousand entries in the most recent miRBase database v21 (Griffiths-Jones et al., 2008; Kozomara and Griffiths-Jones, 2014). Each miRNA can target hundreds of messenger RNAs (mRNAs) in varied binding sites through up- and down- regulation (Ambros, 2004; Thomson and Dinger, 2016). The need for miRNA is usually evidenced by evolutionary conservation and by the many biological processes where they are participating, including creation and physiology (Friedman and Burge, 2014; Skalsky et al., 2014; Taylor et al., 2014). Animal miRNA features,such as developmental timing, neuronal cell fate, cell proliferation and differentiation, rate of metabolism, ageing, apoptosis and organ morphogenesis, suggest that miRNAs are specifically significant for the control of creature birth and loss of life (Ameres and Zamore, 2013; Sun and Lai, 2013). In creature species, sexual dimorphism is normally universal. The important function of the gonads is definitely to synthesize gametes and sex hormones. The sex-biased expression of miRNAs may have got a significant purpose in gonadal differentiation. The major molecular players involved with sexual differential expression have been reported in some non-avian species, such as fruit fly (Marco, 2014), ovine (Torley et al., 2011), porcine (Li et al., 2011b), tilapia (Tao et al., 2016) and yellowish catfish (Jing et al., 2014).

In birds, previous studies on microRNAs mainly centered on the potential miRNAs which could regulate the germ cells and feminine gonads. Firstly, a growing number of miRNAs in birds were identified. For example, Darnell et al (2006) (Darnell et al., 2006) recognized 84 miRNAs in 0.5-5- day-old poultry embryos, Shao et al(Darnell et al., 2006)found 140 potential miRNA loci and 29 novel miRNAs in poultry embryos at eight developmental stages, Glazov et al (2008) (Glazov et al., 2008)discovered 449 innovative chicken miRNAs in chicken embryo, Hicks et al (2008) (Hicks et al., 2008) detected 160 miRNAs (14 are novel) in the embryonic chicken, and Wu et al (2016) identified 663 miRNAs in breeder cock testes. The latest launch (v21) of miRBase reports a complete of 1328 mature miRNAs in birds. On the other hand, miRNAs in the vast majority of birds have not really been reported to day, more sampling is urgently needed to be investigated. Moreover, some essential avian miRNAs involved in regulating gonadal sex differentiation and development had been demonstrated. Bannister et al (2009) (Bannister et al., 2009) advised that MIR202* of chicken may play a critical role in regulating testicular development. Cutting et al (2012) (Cutting et al., 2012) noticed that miR-101, miR-31 and miR-202-5p of chicken show roles testicular and ovarian advancement. More recently, several research on differentially expressed miRNAs (DEMs) in birds have also been reported. Yu et al (2013) (YU et al., 2013) found 55 DEMs between the ovaries of laying and non-laying ducks. Xu et al (2014) (Xu et al., 2014) detected 353 DEMs between that of laying and broody geese. Kang et al (2013) (Kang et al., 2013) discovered 93 DEMs between that of s mature and immature chickens. However, analyses on avian sex-biased expression of miRNAs between for beginners inquiring how to write a high school essay ovaries and testes will be limited.

Parrots, comprising the parakeets, cockatiels, macaws and cockatoos, are members of the Psittciformes buy. There are 397 currently recogniazed species and 94 genera within the order Psittaciformes (Gill and Donsker, 2017). This buy is categorized into four family members: Strigopidae (latest Zealand parrots), Cacatuidae (Cockatoos), Psittacidae (African & New world how to write a conclusion for a research paper parrots), and Psittaculidae (Old community parrots). Parrots, along with corvids, chimps, dolphins, and humans are generally named the most impressive intelligent family pets. The budgerigar (Melopsittacus undulatus) is a little parrot from Australia and a popular pet bird in lots of parts of the world (Collar, 2016) . This bird is the most generally studied parrot species which will serve as an excellent model of behavior and neuroscience. Different studies discovered that vocal learning in adult budgerigars is normally sex-biased (Striedter et al., 2003). In previous research, the budgerigars have already been widely applied in learning vocal learning and sexually dimorphic expansion of males and females (Marisa and Bowling, 2016), however, biological functions of gonadal miRNAs in budgerigars are largely unknown. Moreover, up to now, the sexual differential expression profile of the gonadal miRNAs in budgerigars has not been reported. In today’s study, we performed the expression profiles of miRNAs in two groups of budgerigars (man testes and feminine ovaries) applying illumina paired-end sequencing technology. Based on the new data produced from M. undulatus and existing data from miRBase, we tried to handle (1) identification of noted miRNAs and novel miRNAs in M. undulates, (2) the nucleotide bias of M. undulates miRNAs, (3) Identification of ovary and testis tissues differentially expressed miRNAsin M. undulates, and (4) phylogenetics analysis ofmicroRNA precursor.

  1. Materials and methods

2.1 Ethics statement

Animals in the current study were authorized by the Ethics Committee of Anhui Regular University (Anhui, China) with authorization number #20150612 and humanely sacrificed to ameliorate their suffering as required.

2.2 Tissue collection, RNA planning and sequencing

Six adult 1.5-year-old budgerigars (three males and three females) were obtained from Wuhu (31°33’N, 118°37’E,southeast of China) in 2015. Total RNAs from six gonadal samples had been extracted employing TRIzol reagent (Invitrogen, USA), respectively. The product quality and focus of RNAs had been examined using a ND-8000 spectrophotometer (Nanodrop, Systems, Wilmington, DE) and a 2100-Bioanalyzer (Agilent Technologies, Santa Clara, CA). Two RNA pools for deep sequencing were formed using equal amounts of the extracted RNA from three ovaries and three testes, respectively. The Truseq Compact RNA Sample Preparation Package (Illumina, San Diego, CA) was employed to actually isolate small RNA to construct miRNA library based on the user guide. The resulting cDNA goods were sequenced using the Illumina Hiseq 2500 sequencer (Illumina Inc, San Diego, CA, USA) applying the manual.