Plant Science Today

ashomics@gmail.com Abstract Simple sequence repeats (SSRs) consist of short repeat motifs of 1-6 nucleotides and are found in DNA sequences.The present study was conducted to detect SSRs in chloroplast genome of Tetraphis pellucida (Accession number: NC_024291), downloaded from the National Center for Biotechnology Information (NCBI). The sequence was mined with the help of MISA, a Perl script, to detect SSRs. The length of SSRs defined as ≥12 for mono, di, tri and tetranucleotide, ≥15 for pentanucleotide and ≥18 for hexanucleotide repeats. In total, 41 perfect microsatellites were identified in 127.489 kb sequence mined. An average length of 13.56 bp was calculated for mined SSRs with a density of 1 SSR/3.04 kb. Depending on the repeat units, the length of SSRs ranged from 12 to 20 nt. Dinucleotides (14, 34.15%) were the most frequent repeat type, followed by tetranucleotides (10, 24.39%), trinucleotides (7, 17.07%), mononucleotides (6, 14.63%) and pentanucleotide (4, 9.76%) repeats. Hexanucleotide repeats were completely absent in chloroplast genome of Tetraphis pellucida. The mined SSRs can be used to develop molecular markers and genetic diversity studies in Tetraphis species.


Introduction
Bryophytes are the earliest and the simplest land plants with extant lineages of hornworts, liverworts, and mosses.Paraphyletic origin of bryophytes has been suggested by chloroplast and mitochondrial genome sequences based phylogenetic analysis (Shanker, 2013a;2013b;2013c).Chloroplasts are present in green plants as an intracellular organelles which contain their own autonomously replicating genome.In the recent past organelle genome sequences have been used for various purposes, including the mining of simple sequence repeats (Kapil et al., 2014;Kumar et al., 2014).
Simple sequence repeats (SSRs) also known as microsatellites consist of short repeat motifs (1-6 nucleotides) and are found in both coding and non-coding regions of DNA sequences (Shanker et al., 2007).These repeats act as molecular markers in many plant genomes and also identified in bryophytes (Shanker, 2014a;2014b;2014c;Pandey et al., 2016).However, the distribution of SSRs in chloroplast genome of Tetraphis pellucida Hedw.(Bryophyta), a fourtoothed moss is not well studied.

Tetraphis pellucida: Chloroplast genome sequence
The organelle genome resources at National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov)contain only a handful of chloroplast genome sequences of bryophytes (Shanker, 2012;Shanker, 2015).Recently, Bell et al. (2014) published the chloroplast genome sequence of Tetraphis pellucida.It was downloaded from NCBI in FASTA and GenBank format (NC_024291, 127489bp).

Mining of simple sequence repeats
The chloroplast genome sequence of Tetraphis pellucida was mined using MISA, a Perl script (available at http://pgrc.ipk-gatersleben.de/misa/misa).SSRs length was considered as ≥12 for mono, di, tri and tetranucleotide, ≥15 for pentanucleotide and ≥18 for hexanucleotide repeats.The mined SSRs were classified into coding and non-coding SSRs using the information available in GenBank file of the chloroplast genome.

Results and Discussion
In this study perfect chloroplastic microsatellites or SSRs (cpSSRs) with a minimum length of 12 bp were identified in chloroplast genome sequence of T. pellucida.The length of the identified SSRs ranged from 12 to 20nt.Hexanucleotide repeats were not detected in chloroplast genome sequence of T. pellucida.The distribution of mined SSRs is presented in Fig. 1.
Similar to the cpSSRs in chloroplast genomes of other bryophytes (Shanker 2013d;2013e;Shanker 2014b;2014c) most of the cpSSRs identified in T. pellucida also lie in non-coding region.These cpSSRs can be useful to develop SSR markers and for other purposes.The exclusive genomic and morphological characteristics of Tetraphis authenticate its significance for deciding one of the key issues in the phylogeny of land plant and for understanding the evolution of the some special structures.Hence, this study provides a scientific base for phylogenetics, evolutionary genetics and diversity studies on different Tetraphis species.

Figure 1 .
Figure 1.Frequency distribution of various repeat types