Characteristics of SSR Markers in Floccularia Luteo-virens by RNA -Sequencing

Simple sequence repeats are short tandem repeats; SSRs are widespread in both coding and non-coding regions of prokaryotic and eukaryotic genomes with a high level of polymorphism [1]. Because of their high polymorphism, abundance, and co dominant inheritance, SSRs are well suited for the assessment of genetic diversity within crop species and the genetic relationships among species [2]. Floccularia luteo-virens, which is also referred to as yellow mushroom and belongs to Basidiomycotina, Hymenomycetes, Agaricales, Tricholomataceae, is known as one of the most appreciated wild mushrooms in China. The mushroom is an expensive edible medical fungus, rich in protein, minerals, amino acids and multivitamins. It has anti-influenza, anti-neuritis, promoting children’s development, anti-cancer and other pharmacological functions [3]. In recent years, the growth area of Floccularia luteo-virens in alpine meadow has been decreasing at a high rate [4], and as a consequence, Floccularia luteo-virens becomes increasingly less available. Knowledge of the characteristics of SSR markers in Floccularia luteo-virens helps to mitigate the potential damage to Floccularia luteo-virens and protect biodiversity.


Introduction
Simple sequence repeats are short tandem repeats; SSRs are widespread in both coding and non-coding regions of prokaryotic and eukaryotic genomes with a high level of polymorphism [1]. Because of their high polymorphism, abundance, and co dominant inheritance, SSRs are well suited for the assessment of genetic diversity within crop species and the genetic relationships among species [2]. Floccularia luteo-virens, which is also referred to as yellow mushroom and belongs to Basidiomycotina, Hymenomycetes, Agaricales, Tricholomataceae, is known as one of the most appreciated wild mushrooms in China. The mushroom is an expensive edible medical fungus, rich in protein, minerals, amino acids and multivitamins. It has anti-influenza, anti-neuritis, promoting children's development, anti-cancer and other pharmacological functions [3]. In recent years, the growth area of Floccularia luteo-virens in alpine meadow has been decreasing at a high rate [4], and as a consequence, Floccularia luteo-virens becomes increasingly less available. Knowledge of the characteristics of SSR markers in Floccularia luteo-virens helps to mitigate the potential damage to Floccularia luteo-virens and protect biodiversity.

Fungal Materials
The fruiting bodies of Floccularia luteo-virens in the juvenile or adult stage were collected from their natural environment in Haiyan County, Qinghai Province, China, in July, 2015. The juvenile stage is defined as the stage in which the cap diameter of Floccularia luteo-virens is ≦5cm; while the adult stage is defined as the stage in which the cap diameter of Floccularia luteo-virens is > 5cm.

Extraction of Total RNA
Fresh tissues (50mg) were obtained from the cap and stipe of the fruiting bodies using a single blade for the extraction of total RNA following the manufacturer's instructions.

Determination of The Purity and Concentration of RNA
The purity and concentration of total RNA were determined using a nucleic acid and protein analyzer. The contamination of genomic DNA was determined by 1% agarose gel electrophoresis.

RNA-Seq
The total RNA samples obtained from the fruiting bodies of Floccularia luteo-virens in the juvenile or adult stage were sent to Sangon Biotech (Shanghai, China) for transcriptome sequencing. RNA-seq was performed using the Solexa Hiseq 2500 highthroughput sequencing platform (Illumina, USA) by the PE125 strategy. The sequences of the two samples were merged and de novo assembled. Repeated sequences were removed, and the sequences of > 200 bp were selected. The longest transcript of each locus was used for Unigene. The identification and localization of SSRs were performed using MISA. The density of different motifs and repeat number in Transcript and Unigene were determined. The primers of SSR-PCR were designed by Primer 3.
The SSRs with ≤11 repeats accounted for 96.73% of the total SSRs. For Transcript, the SSRs found in this study could be categorized by 100 types of motifs (without considering sequence complementary). In general, SSRs were unevenly distributed across motif types. Motif A had the highest frequency of 17.50% in mononucleotides, followed by motif GT (7.83%) in dinucleotides and motif CAA (1.53%) in trinucleotides respectively, and the ratio of mononucleotides was A>T>G>C. But for Unigene, the SSRs found in this study could be categorized by 98 types of motifs. In general, SSRs were also unevenly distributed across motif types. Motif A had the highest frequency of 11.65% in mononucleotides, followed by motif TG in dinucleotides (10.86%) and motif TGT (1.37%) in trinucleotides, and the ratio of dinucleotides was TG/GT>AC/ CA>AT/TA>AG/GA (considering sequence complementary).

Discussion
SSR technology is one of the most widely used molecular markers, it has been widely used in all kinds of organisms. However, there have been few studies on the development and application of SSR markers in Floccularia luteo-virens. Thus, our study may provide some insights into the application of SSR molecular markers in Floccularia luteo-virens.
In this study, we found that dinucleotide repeats were the most abundant SSR type in Unigene of Floccularia luteo-virens, which was same as that in Actinidia [5]. Moreover, dinucleotide repeats have been reported as the SSR type in ESTs with highest abundance of many animal species, such as Xiphophorus, Fundulus and others [6], and trinucleotide repeats have been reported as the SSR type with highest abundance in plants [7,8].