Analysis of Simple Sequence Repeats (SSRs) Dynamics in Reticulitermes chinensis

: An abundance of potential inherent variation in SSR or microsatellite repeats has resulted in a valuable genetic marker in eukaryotes. We describe how SSRs in Reticulitermes chinensis castes are organized and abundant. Collectively we sequence 184,436 unigenes through the Trinity system, ranging from 201-43, 214 bp length from transcriptome data. Using MISA to find out SSRs in uni-genes and a total of 10740 SSRs loci were identified as di-, tri-, tetra-, penta-, and hexanucleotide. Among them, trinucleotide SSRs were the most abundant in R. chinensis genome 2702, followed by chromosome six (tri-) 1110. Pentanucleotide repeats were found at frequency 1 from each chromosome 8, 9 and 13, respectively. The frequency of AC/GT motif was (21.91%) reported, followed by others motif (16.6%), AAG/CTT (8.49%) and AGC/CTG (8.2%). The minimum motif types were AATG/ATTC (1.27%), followed by ACG/CGT (1.32%) and AAT/ATT (1.77%). Thes abundance and inherent variations in SSRs provide valuable information for taxonomics, phylogenetic, genome mapping, and population genetic research studies. SSR-based markers have high degrees of allelic variability and codominant legacy ( inheritance), and analytical ease.


Introduction
Termites are well-known eusocial insects classified into nine families, with 14 subfamilies, 280 types, and more than 2600 species [1].Approximately 4,000 species are estimated (only 2,600 are taxonomically known).It should be noted that only about 10% are considered pests.Termites are significant detritivores, especially in the subtropical and tropical regions, and it is considered ecological important to recycle wood and other plant materials [2].Termite colonies depended entirely on tissues intact or partially decayed of wood, whether living or dead or mass of plants.It is becoming an economic pest when lumber products, construction materials, forests, and other commercial products begin to be destroyed [3].Termite has diverse feeding behavior consuming wood cellulose from tree farming, structural lumber, and causes enormous economic losses up to $40 billion worldwide/year [4,5].In China, more than 1.8 billion RMB is used to control Reticulitermes aculabialis, R. chinensis, R. labralis, R. speratus, and R. flaviceps termites species [3].Annual losses were estimated to 0. steadily increasing, even with management strategies, and cause enormous economic causes to the Chinese economy annually [6,7].Microsatellite markers or simple sequence repeats (SSRs) are the best-known and influential tools that have emerged for determining genetic divergence among populations, structure, and genetic diversity estimates, among different taxa [8][9][10], which is critical to the development of efficient conservation strategies.In estimating possible annihilation caused in individual natural populations, microsatellites as an advanced tool are fundamentally important.In addition to the structural situation of genetic composition, microsatellites can also assess reproductive behavior, social structure, and dispersal of endangered insect species [11,12].However, this approach is desirable to the generation of microsatellite markers because of low costs of in silico mining and large abundance of microsatellites in different sequence resources.The negligible costs of in silico mining and many microsatellites in various sequence resources make this approach particularly attractive for creating microsatellite markers.Due to its very polymorphic nature, SSR offers a powerful taxonomic, phylogenetic, and population genetic studies tool.Polymorphism in SSRs generally considered slippage and unequal recombination of DNA polymerase [12][13][14].The abundance and distribution of SSRs helped to understand the relevance of genes or genome development.The current study aimed to analyze transcriptomes of Reticulitermes chinensis, and distribution of various SSR loci were in silico identified from transcriptome unigenes, which could help to understand the evolutionary consequences and analysis of diversity.

Transcriptome Sequencing
Reticulitermes chinensis colonies were collected from Qinling mountains and reared at room temperature during April 2014-May 2018, Northwest University, Xian, China (http://english.nwu.edu.cn/).An adequate amount of RNA was obtained from PKs (primary king), PQs (primary queen) [15], ergatoid (SWRK "secondary worker reproductive king and SWRQ "queen"), WM (male workers) and WF (female workers), for Illumina sequencing using TRIzol reagent and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) [16].The total amount of RNA was extracted and stored at −80°C for further experiments.Later, NEB-Next Prep-Kit for Illumina (NEB) sequences [17] was used to reverted cDNA according to the manufacturer protocol.A spectrophotometer was used for the quantity and quality of RNA and cDNA.Three technical and five biological replicates were pooled for each caste and stored at −20 °C for further experiments.

Simple sequence repeat (SSR) analysis
We annotated sequence reads through Illumina HiSeqTM 4000 platform and Trinity system (trinityrnaseq r2012-04-27), ranging from various bp length at transcriptome data [15].The estimated GC performance evaluation and assessment were estimated as exemplary quality assemblies for all unigenes in the transcriptome data utilized MISA (http:/pgrc.ipk-gatersleben.de/misa/) to find out SSR in unigenes [18].The predicted and identified SSRs were di-, tri-, tetra-, penta-, and hexanucleotide motifs with amplification products containing repeat units ≥6 times.

Discussions
In research on genetic diversity, genetic mapping, comparative genomics, and marker-assisted selection breeding, polymorphic SSRs play an essential role.Transcriptomics provides a rich discovery source of SSR because it creates many sequences [19].The transcriptomes analysis of R. chinensis was reported annotated 103589264 clean assembled sequence reads (561 average bp length) via the Illumina HiSeqTM 4000 platform.A total of 184,436 unigenes were assembled (201 to 43,214bp) from accurate transcriptome data via the Trinity system (7G).We estimated that 43.02% of GC performance evaluation and assessment suggests that quality and development in sequence are appropriate for homogenous studies.
The transcriptome-SSR frequency has several factors.The first factor of the transcriptome-SSR frequency, e.g., repeat length threshold and several repetition motifs, are called microsatellite criteria [20,21].Most studies excluded repeat motifs of mononucleotides, as these could result from sequencing errors.In some studies, the number of dinucleotide reproductions based on three repeat units, while others are less [22][23][24].Moreover, SSRs were mainly identified from unigenes above 1000 bp, reducing the frequency to some extent.Second, SSRs frequency may also be affected by genome structure or composition [25].For instance, Reticulitermes small genome size was reported as the cause of the high frequency of transcriptomes-SSR sequences [15,26].Finally, the various SSR software can also affect the frequency of SSRs loci.In addition to genomic stability, the effects of excess numbers of short-iterate repeats might significantly develop genomic characteristics, such as codon usage [13].Microsatellites generally show an increase in repeat length [27] to decrease in abundance.However, there were also reports of more than expected long microsatellite replicates [28].For the development of specific genome markers, the microsatellites identified in this study could be used in evolutionary studies in Reticulitermes castes.

Conclusion
In addition to the advanced computational field, the comprehensive review has shown development in identifying an SSRs motif in the R. chinensis.With the development of sequencing methodologies, massive sequence data for identification and quantification of SSRs motif in the R. chinensis genome has been generated in transcriptome data.The results of reliable next-generation sequencing (NGS) technologies with fast and cost-effective methods will continue to provide additional information on SSR markers in insects.Thus, in the new-model R. chinensis field, including evolutionary consequences and the analysis of diversity to improve genetically, the NGS area added advantage in the SSR marker development.The overall conclusion of this study is to provide valuable information for the SSR motif of R. chinensis and future genetic or genomic studies in economically significant (detritivores the plants and buildings) valuable.

Supplementary Materials:
The following are available online at www.mdpi.com/xxx/s1,Table S1: Total of annotated genes with sequenced genes in different castes of R. chinensis.Table S2: The total number of unigenes, unmapped, unique, multiple, and total mapped unigenes in R. chinensis castes.Table S3: Distribution of SSRs in different chromosomes identified from transcriptomes of R. chinensis.Table S4: Motif and their frequency (%) from transcriptomes data in R. chinensis.
Funding: Financial support was provided by the National Natural Science Foundation of China (31870389) and Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education (ZSK2017002).

Informed Consent Statement: Not applicable.
Data Availability Statement: All data and transcriptomic sequences will be available under BioProject accession number PRJNA592596 at the NCBI.The corresponding author will answer any reasonable requests.

Conflicts of Interest:
The authors declare no conflict of interest.

Ethical approval:
Reticulitermes chinensis species is a common species; therefore, no ethical approval required.
3 billion dollars in 2004, and approximately 217 million dollars a year cost the wood damage alone by this pest.Termites affect more than 90% of homes south of the Yangtze River.The damage and economic losses caused by termites are Citation: Haroon; Li, Y.-X.; Ye, C.-X.; Ma, X.-Q.; Su, J.; Su, X.-H.; Xing, L.X. Analysis of Simple Sequence Repeats (SSRs) Dynamics in Reticulitermes chinensis, in Proceedings of the 1st International Electronic Conference on Entomology, 1-15 July 2021, MDPI: Basel, Switzerland, doi:10.3390/IECE-10566Published: 2 July 2021 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.Copyright: © 2021 by the authors.Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses /by/4.0/).

Figure 1 .
Figure 1.The total number of genes and sequence genes from R. chinensis.

Figure 4 .
Figure 4. Frequency distribution of SSRs according to motif and sequence types.

Table S1 .
Total of annotated genes with sequenced genes in different castes of R.

Table S3 .
Distribution of SSRs in different chromosomes identified from transcriptomes of R. chinensis.

Table S4 .
Motif and their frequency (%) from transcriptomes data in R. chinensis.