Perfect Simple Sequence Repeats Research Articles

Identification and characterization of novel drought-responsive lncRNAs in stone apple (Aegle marmelos L.) through whole-transcriptome analysis

Stone apple (Aegle marmelos L.) is a subtropical fruit tree of the Rutaceae family, highly valued in traditional medicine across the Indian subcontinent. We conceived this study with the objective of developing a comprehensive transcriptome dataset, identifying SSRs for marker-assisted breeding, and delineating regulators of gene expression, with a specific emphasis on non-coding RNA (ncRNA), particularly related to drought stress. To achieve this, RNA-seq was conducted using RNA pooled from various tissues, including roots, leaves, inflorescence, and developing seeds from stone apple, and the clean reads were assembled into 40,886 unigenes. Subsequently, the unigenes were categorized into gene ontology categories encompassing biological processes, molecular functions, and cellular components. Within the unigenes, we identified a total of 9174 perfect simple sequence repeats (SSRs), 2167 transcription factors (TFs) distributed among 69 families, and 415 transcription regulators (TRs) across 27 families. Additionally, 19 microRNAs (miRNAs) from 12 families, 16,811 potential long noncoding RNAs (lncRNAs), and six functional endogenous target mimics (eTMs) were detected. Analysis of lncRNA-miRNA-mRNA interactions unveiled multiple regulatory nodes, elucidating lncRNA/miRNA-driven gene expression control in stone apple. The increased co-expression of selected drought-related lncRNAs and their cognate target mRNAs supported the aforementioned findings under drought conditions. Overall, this study significantly advances our understanding of stone apple genomics and lays a foundation for future omics-based studies, thereby facilitating the deployment of climate-resilient strategies in the species.

Investigating the patterns of simple sequence repeats (SSRs) inferred from the whole genome of a critically endangered species Cassine koordersii kosterm

Cassine koordersii Kosterm. (Celastraceae) is a critically endangered species indigenous to Jember, East Java. Programs for genetic conservation and plant breeding have recently implemented next-generation sequencing (NGS) techniques based on genomic data. This research aims to explore and distinguish between perfect and imperfect SSR patterns in the assembled genome. The Abyss assembler produced 3,060,362 scaffolds with 35.63 % GC content for the assembled genome. The investigation and identification of SSRs using the Krait tool found 139,236 and 582,360 sequences for including perfect and imperfect SSRs, respectively. There were six motif repeats of perfect and imperfect SSRs consisting of 73,175 and 202,438 sequences of mononucleotide (the most motif was A); 17,179 and 65,705 sequences of dinucleotide (the most motif was AT); 5,175 and 51,948 sequences of trinucleotide (the most motif was AAT); 3,824 and 14,010 sequences of tetranucleotide (the most motif was AAAT); 659 and 3,082 sequences of pentanucleotide (the most motif is AAAAT); 118 and 757 sequences of hexanucleotide (the most motif is AAAAAT). The depicted perfect and imperfect SSRs markers can be employed in future genetic studies of Cassine and related genera for either recommendation effort or improvement in conservation genetic concerns.

Comprehensive genome-wide identification and transferability of chromosome-specific highly variable microsatellite markers from citrus species

Citrus species among the most important and widely consumed fruit in the world due to Vitamin C, essential oil glands, and flavonoids. Highly variable simple sequence repeats (SSR) markers are one of the most informative and versatile molecular markers used in perennial tree genetic research. SSR survey of Citrus sinensis and Citrus maxima were identified perfect SSRs spanning nine chromosomes. Furthermore, we categorized all SSR motifs into three major classes based on their tract lengths. We designed and validated a class I SSRs in the C. sinensis and C. maxima genome through electronic polymerase chain reaction (ePCR) and found 83.89% in C. sinensis and 78.52% in C. maxima SSRs producing a single amplicon. Then, we selected extremely variable SSRs (> 40 nt) from the ePCR-verified class I SSRs and in silico validated across seven draft genomes of citrus, which provided us a subset of 84.74% in C. sinensis and 77.53% in C. maxima highly polymorphic SSRs. Out of these, 129 primers were validated on 24 citrus genotypes through wet-lab experiment. We found 127 (98.45%) polymorphic HvSSRs on 24 genotypes. The utility of the developed HvSSRs was demonstrated by analysing genetic diversity of 181 citrus genotypes using 17 HvSSRs spanning nine citrus chromosomes and were divided into 11 main groups through 17 HvSSRs. These chromosome-specific SSRs will serve as a powerful genomic tool used for future QTL mapping, molecular breeding, investigation of population genetic diversity, comparative mapping, and evolutionary studies among citrus and other relative genera/species.

Small cardamom genome: development and utilization of microsatellite markers from a draft genome sequence of Elettaria cardamomum Maton.

Small cardamom (Elettaria cardamomum Maton), the queen of spices, is the third most expensive spice in the world after saffron and vanilla, valued highly for its aroma and taste. This perennial herbaceous plant is a native of coastal parts of Southern India and displays a significant amount of morphological diversity. Its genetic potential has not been exploited due to lack of genomic resources limiting our understanding of the genome and important metabolic pathways which give it the economic advantage in the spice industry. Here, we report upon the de novo assembled, draft whole genome sequence of cardamom variety, Njallani Green Gold. We used a hybrid assembly strategy using the reads from the Oxford Nanopore, Illumina and 10x Genomics GemCode sequencing chemistries. The assembled genome length was 1.06 Gb (gigabases) which is close to the estimated genome size of cardamom. More than 75% of the genome was captured in 8000 scaffolds with a N50 of 0.15 Mb. The genome appears to have a high repeat content and 68055 gene models were predicted. The genome is close to Musa species and displays an expansion and contraction in different gene families. The draft assembly was used for in silico mining of simple sequence repeats (SSRs). A total of 2,50,571 SSRs were identified of which 2,18,270 were perfect SSRs and 32,301 were compound SSRs. Among the perfect SSRs, trinucleotides were most abundant (1,25,329) and hexanucleotide repeats appear least (2,380). From the 2,50,571 SSRs mined, 2,27,808 primer pairs were designed based on flanking sequence information. Wet lab validation was performed for 246 SSR loci and based on their amplification profiles, 60 SSR markers were used for diversity analysis of a set of 60 diverse cardamom accessions. The average number of alleles detected per locus were 14.57 with a minimum of 4 and maximum of 30 alleles. Population structure analysis revealed the presence of high degree of admixtures which could primarily be due to cross-pollination prevalent in this species. The SSR markers identified would help in the development of gene or trait-linked markers which can be subsequently used for marker-assisted breeding for crop improvement in cardamom. The information on utilization of the SSR loci for generation of markers has been developed into a public database, 'cardamomSSRdb' that is freely available for use by the cardamom community.

Genetic diversity analysis and variety identification using SSR and SNP markers in melon

Melon is an important horticultural crop with a pleasant aromatic flavor and abundance of health-promoting substances. Numerous melon varieties have been cultivated worldwide in recent years, but the high number of varieties and the high similarity between them poses a major challenge for variety evaluation, discrimination, as well as innovation in breeding. Recently, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), two robust molecular markers, have been utilized as a rapid and reliable method for variety identification. To elucidate the genetic structure and diversity of melon varieties, we screened out 136 perfect SSRs and 164 perfect SNPs from the resequencing data of 149 accessions, including the most representative lines worldwide. This study established the DNA fingerprint of 259 widely-cultivated melon varieties in China using Target-seq technology. All melon varieties were classified into five subgruops, including ssp. agrestis, ssp. melo, muskmelon and two subgroups of foreign individuals. Compared with ssp. melo, the ssp. agrestis varieties might be exposed to a high risk of genetic erosion due to their extremely narrow genetic background. Increasing the gene exchange between ssp. melo and ssp. agrestis is therefore necessary in the breeding procedure. In addition, analysis of the DNA fingerprints of the 259 melon varieties showed a good linear correlation (R2 = 0.9722) between the SSR genotyping and SNP genotyping methods in variety identification. The pedigree analysis based on the DNA fingerprint of ‘Jingyu’ and ‘Jingmi’ series melon varieties was consistent with their breeding history. Based on the SNP index analysis, ssp. agrestis had low gene exchange with ssp. melo in chromosome 4, 7, 10, 11and 12, two specific SNP loci were verified to distinguish ssp. agrestis and ssp. melon varieties. Finally, 23 SSRs and 40 SNPs were selected as the core sets of markers for application in variety identification, which could be efficiently applied to variety authentication, variety monitoring, as well as the protection of intellectual property rights in melon.

Genetic structure of Spirometra mansoni (Cestoda: Diphyllobothriidae) populations in China revealed by a Target SSR-seq method

BackgroundIn China, the plerocercoid of the cestode Spirometra mansoni is the main causative agent of human and animal sparganosis. However, the population genetic structure of this parasite remains unclear. In this study, we genotyped S. mansoni isolates with the aim to improve current knowledge on the evolution and population diversity of this cestode.MethodsWe first screened 34 perfect simple sequence repeats (SSRs) using all available omic data and then constructed target sequencing technology (Target SSR-seq) based on the Illumina NovaSeq platform. Next, a series of STRUCTURE. clustering, principal component, analysis of molecular variance and TreeMix analyses were performed on 362 worm samples isolated from 12 different hosts in 16 geographical populations of China to identify the genetic structure.ResultsA total of 170 alleles were detected. The whole population could be organized and was found to be derived from the admixture of two ancestral clusters. TreeMix analysis hinted that possible gene flow occurred from Guizhou (GZ) to Sichuan (SC), SC to Jaingxi (JX), SC to Hubei (HB), GZ to Yunnan (YN) and GZ to Jiangsu (JS). Both neighbor-joining clustering and principal coordinate analysis showed that isolates from intermediate hosts tend to cluster together, while parasites from definitive hosts revealed greater genetic differences. Generally, a S. mansoni population was observed to harbor high genetic diversity, moderate genetic differentiation and a little genetic exchange among geographical populations.ConclusionsA Target SSR-seq genotyping method was successfully developed, and an in-depth view of genetic diversity and genetic relationship will have important implications for the prevention and control of sparganosis.Graphical

Evaluation of Perfect Microsatellites in Nile Tilapia (Oreochromis niloticus) Genome

Microsatellites or simple sequence repeats (SSRs) consist of a sizable part of genomes and play a crucial role in the function of genes and the organization of the genome. The complete availability of a genome sequence for Nile tilapia (Oreochromis niloticus) provides the possibility of accomplishing a genome-wide analysis of SSRs in this species. I analyzed the abundance and density of perfect SSRs in the Nile tilapia genome and observed a sum of 252,047 microsatellites with 1–6 bp nucleotide motifs. This indicates that about 2.7 % of the Nile tilapia whole genome sequence (927.77Mb) is made up of perfect SSRs, with an average length of 135.68bp/Mb. The average density and frequency of perfect SSRs were 271.69 loci/Mb and 5834.46 bp/Mb, respectively. The six classes of perfect SSRs proportional distribution within the Nile tilapia genome were not even. Dinucleotide repeats (40.13 %) with a total count of 101145 of an average length of 26.11 bp happen to be the most abundant class of SSRs, while the percentages of mononucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats was 31.88 %, 11.98 %, 11.52 %, 4.22 %, and 0.26 %, accordingly. The various classes of SSRs repeat differ in their number of repeats with the highest being 95. My results indicate that 21 motifs contain the prevalent categories with a frequency above 1 locus/Mb: A, AAC, AAG, AATAG, AATTC, AC, AG, AGAT, AT, ATCT, ATG, ATGG, ATT, ATTT, C, CCT, CTG, CTTT, GT, GTTT.

Mining and analysis of microsatellites in human coronavirus genomes using the in-house built Java pipeline.

Microsatellites or simple sequence repeats are motifs of 1 to 6 nucleotides in length present in both coding and non-coding regions of DNA. These are found widely distributed in the whole genome of prokaryotes, eukaryotes, bacteria, and viruses and are used as molecular markers in studying DNA variations, gene regulation, genetic diversity and evolutionary studies, etc. However, in vitro microsatellite identification proves to be time-consuming and expensive. Therefore, the present research has been focused on using an in-house built java pipeline to identify, analyse, design primers and find related statistics of perfect and compound microsatellites in the seven complete genome sequences of coronavirus, including the genome of coronavirus disease 2019, where the host is Homo sapiens. Based on search criteria among seven genomic sequences, it was revealed that the total number of perfect simple sequence repeats (SSRs) found to be in the range of 76 to 118 and compound SSRs from 01 to10, thus reflecting the low conversion of perfect simple sequence to compound repeats. Furthermore, the incidence of SSRs was insignificant but positively correlated with genome size (R2 = 0.45, p > 0.05), with simple sequence repeats relative abundance (R2 = 0.18, p > 0.05) and relative density (R2 = 0.23, p > 0.05). Dinucleotide repeats were the most abundant in the coding region of the genome, followed by tri, mono, and tetra. This comparative study would help us understand the evolutionary relationship, genetic diversity, and hypervariability in minimal time and cost.

Microsatellite Variation in the Most Devastating Beetle Pests (Coleoptera: Curculionidae) of Agricultural and Forest Crops.

Weevils, classified in the family Curculionidae (true weevils), constitute a group of phytophagous insects of which many species are considered significant pests of crops. Within this family, the red palm weevil (RPW), Rhynchophorus ferrugineus, has an integral role in destroying crops and has invaded all countries of the Middle East and many in North Africa, Southern Europe, Southeast Asia, Oceania, and the Caribbean Islands. Simple sequence repeats (SSRs), also termed microsatellites, have become the DNA marker technology most applied to study population structure, evolution, and genetic diversity. Although these markers have been widely examined in many mammalian and plant species, and draft genome assemblies are available for many species of true weevils, very little is yet known about SSRs in weevil genomes. Here we carried out a comparative analysis examining and comparing the relative abundance, relative density, and GC content of SSRs in previously sequenced draft genomes of nine true weevils, with an emphasis on R. ferrugineus. We also used Illumina paired-end sequencing to generate draft sequence for adult female RPW and characterized it in terms of perfect SSRs with 1–6 bp nucleotide motifs. Among weevil genomes, mono- to trinucleotide SSRs were the most frequent, and mono-, di-, and hexanucleotide SSRs exhibited the highest GC content. In these draft genomes, SSR number and genome size were significantly correlated. This work will aid our understanding of the genome architecture and evolution of Curculionidae weevils and facilitate exploring SSR molecular marker development in these species.

Comprehensive Analyses of Simple Sequence Repeat (SSR) in Bamboo Genomes and Development of SSR Markers with Peroxidase Genes.

Simple sequence repeats (SSRs) are one of the most important molecular markers, which are widespread in plants. Bamboos are important forest resources worldwide. Here, the comprehensive identification and comparative analysis of SSRs were performed in three woody and two herbaceous bamboo species. Altogether 567,175 perfect SSRs and 71,141 compound SSRs were identified from 5737.8 Mb genome sequences of five bamboo species. Di-nucleotide SSRs were the most predominant type, with an average of ~50,152.2 per species. Most SSRs were located in intergenic regions, while those located in genic regions were relatively less. Moreover, the results of annotation distribution indicated that terms with P450, peroxidase and ATP-binding cassette transporter related to lignin biosynthesis might play important roles in woody and herbaceous bamboos under the mediation of SSRs. Furthermore, the peroxidase gene family consisted of a large number of genes containing SSRs was selected for the evolutionary relationship analysis and SSR markers development. Fifteen SSR markers derived from peroxidase family genes of Phyllostachys edulis were identified as polymorphic in 34 accessions belonging to seven genera in Bambusoideae. These results provided a comprehensive insight of SSR markers into bamboo genomes, which would facilitate bamboo research related to comparative genomics, evolution and marker-assisted selection.

TinoTranscriptDB: A Database of Transcripts and Microsatellite Markers of Tinospora cordifolia, an Important Medicinal Plant.

Tinospora cordifolia, commonly known as “Giloe” in India, is a shrub belonging to the family Menispermaceae. It is an important medicinal plant known for its antipyretic, anti-inflammatory, antispasmodic, and antidiabetic properties and is used in the treatment of jaundice, gout, and rheumatism. Despite its economic importance, the limited information related to its genomic resources prohibits its judicious exploitation through molecular breeding or biotechnological approaches. In this study, we generated a meta-transcriptome assembly of 43,090 non-redundant transcripts by merging the RNASeq data obtained from Roche 454 GS-FLX, and Illumina platforms, and report the first transcriptome-based database for simple sequence repeats and transcription factors (“TinoTranscriptDB” (Tinospora cordifolia Transcriptome Database)). We annotated 26,716 (62%) of the total transcripts successfully from National Center for Biotechnology Information non-redundant protein (NCBI-NR), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-Prot, and Pfam databases. This database contains information of 2620 perfect simple sequence repeats (P-SSRs) with a relative abundance of 340.12 (loci/Mb), and relative density of 6309.29 (bp/Mb). Excluding mono-nucleotides, the most abundant SSR motifs were tri-nucleotides (54.31%), followed by di-nucleotides (37.51%), tetra-nucleotides (4.54%), penta-nucleotides (3.16%) and hexa-nucleotides (0.45%). Additionally, we also identified 4,311 transcription factors (TFs) and categorized them into 55 sub-families. This database is expected to fill the gap in genomic resource availability in T. cordifolia and thus accelerate molecular breeding and related functional and other applied studies aimed towards genetic improvements of T. cordifolia and related species.

Development of novel genome‑wide simple sequence repeats (SSR) markers in Bunium persicum

Bunium persicum (Boiss.) Fedtsch or Kala jeera is an economically important medicinal spice that grows in cold temperate regions of Central Asia and Northern India. It is mainly used as spice and condiment in various food preparations. However, its high aroma and richness in essential oils make it industrially important. The bioactivity and pharmacological activities of its phytochemicals have been well elucidated. Due to limited cultivation and huge harvest pressure on natural resources, this species is under severe threat of diversity loss. Further, genetics and molecular breeding studies are lagging for this important crop plant due to a dearth of efficient genome wide molecular markers. In the present study, genomic resources for B. persicum have been generated for the first time through de novo sequencing. In addition, novel genomic SSRs from these resources are identified for their use in future diversity analysis and breeding programs. A total of 177029 perfect and 5915 compound SSR motifs were identified in 212585 assembled sequences. Mononucleotide repeats were the most abundant (55.18%) followed by di- (32.35%), tri- (7.21%), tetra- (3.15%) and hexa- nucleotide (0.76%) repeats. Total 101519 and 2649 primers were successfully designed for sequences containing perfect and compound SSR repeat motifs, respectively. For validation, 88 primer pairs were synthesized for perfect SSR motifs, from which 68 pairs gave scorable amplification. From these, 16 primer pairs were found monomorphic and 52 were polymorphic. These 68 primer pairs altogether yielded 101 alleles with an average of 1.71 alleles per loci. Overall average allele frequency (0.7908), PIC value (0.213), heterozygosity value (0.2995), and gene diversity (0.2680) was recorded for the evaluated Bunium accessions. UPGMA tree constructed based on Nei genetic distances successfully distinguished the evaluated B. persicum accessions. Low genetic diversity recorded in the present study emphasizes the need for restoration and maintenance of effective B. persicum population sizes in situ for its future sustenance.

Expressed sequence tag simple sequence repeats (EST-SSRs) mining and marker development from Leucaena leucocephala root transcriptome

Leucaena leucocephala is a nitrogen-fixing legume and a fast-growing species used for various purposes, including food, medicine, and most recently, energy-source wood. Compared to other commonly planted legumes, this species has a high-stress tolerance, and only one recorded insect infestation. The transcriptome analysis revealed genes involved in the production of stress tolerance compounds in the root. In this study, we attempted to extract simple sequence repeats (SSRs) from the transcriptome data of L. leucocephala root to establish molecular markers. The SSRs mined from L. leucocephala transcriptome deposited in NCBI with accession number GDRZ00000000. We used Krait v.1.3.3 to extract the SSRs and developed the primers. The SSRs cover 0.3% of the total transcriptome sequence, with 21.321 perfect SSRs found and relative density 2908.63 (bp/Mb). The most abundant type of SSRs was found in mononucleotide (39.85%), followed by dinucleotide (33.49%) and trinucleotide (24.75). However, for tetra, penta, and hexanucleotide, the percentage was lower than 2%. The number of primers generated was 8137 primers, with 3969 dinucleotides, 3947 trinucleotides, 150 tetranucleotides, 21 pentanucleotides, and 50 hexanucleotide SSR primers. This finding may help to accelerate stress-tolerant legume improvement in the future.

Development of Genome-wide Simple Sequence Repeat Markers from Whole-genome Sequence of Mungbean (Vigna radiata)

Background: Mungbean is an important pulse crop and it is mainly cultivated in Asia for human consumption. Its small genome and diploid nature make it a well-suited model organism among legume crops. Thus, cost-effective, reliable and highly polymorphic molecular markers distributing the whole genome are needed for diversity, mapping and functional genomics studies in this model species. Methods: The whole-genome sequence of mungbean was obtained and used as a source of identification of simple sequence repeats (SSR). The sequence reads were aligned and SSRs detection was performed using the Phobos plugin tandem repeat finder in the Geneious software program. A total of 12 mungbean genotypes were selected to validate the newly developed SSR markers. Result: In the present study, a total of 12, 49,774 and 11, 86, 386 perfect and imperfect SSR repeats were identified from the mungbean genome. The tri-repeats were the most abundant (26.10%), followed by hexa (20.82%), penta (20.45%), tetra (17.65%) and di-repeats (14.95%). We designed 1330 SSR primers based on the genomic sequence of flanking perfect SSRs (Di and tri-repeats). Among them, 50 SSR primers uniformly distributed across the 11 mungbean chromosomes were selected and used to validate 12 mungbean genotypes. The newly developed genomic SSR markers generated in the present study are a valuable genomic resource for the mungbean breeding programs.

Characterization and complexity of transcriptome in Gymnocypris przewalskii using single-molecule long-read sequencing and RNA-seq

The Tibetan Schizothoracinae fish Gymnocypris przewalskii has the ability to adapt to the extreme plateau environment, making it an ideal biological material for evolutionary biology research. However, the lack of well-annotated reference genomes has limited the study of the molecular genetics of G. przewalskii. To characterize its transcriptome features, we first used long-read sequencing technology in combination with RNA-seq for transcriptomic analysis. A total of 159,053 full-length (FL) transcripts were captured by Iso-Seq, having a mean length of 3,445 bp with N50 value of 4,348. Of all FL transcripts, 145,169 were well-annotated in the public database and 134,537 contained complete open reading frames. There were 4,149 pairs of alternative splicing events, of which three randomly selected were defined by RT–PCR and sequencing, and 13,293 long non-coding RNAs detected, based on all-vs.-all BLAST. A total of 118,185 perfect simple sequence repeats were identified from FL transcripts. The FL transcriptome might provide basis for further research of G. przewalskii.

Comprehensive Characterization and Validation of Chromosome-Specific Highly Polymorphic SSR Markers From Pomegranate (Punica granatum L.) cv. Tunisia Genome.

The simple sequence repeat (SSR) survey of ‘Tunisia’ genome (296.85 Mb) identified a total of 365,279 perfect SSRs spanning eight chromosomes, with a mean marker density of 1,230.6 SSRs/Mb. We found a positive trend in chromosome length and the SSR abundance as marker density enhanced with a shorter chromosome length. The highest number of SSRs (60,708) was mined from chromosome 1 (55.56 Mb), whereas the highest marker density (1,294.62 SSRs/Mb) was recorded for the shortest chromosome 8 (27.99 Mb). Furthermore, we categorized all SSR motifs into three major classes based on their tract lengths. Across the eight chromosomes, the class III had maximum number of SSR motifs (301,684, 82.59%), followed by the class II (31,056, 8.50%) and the class I (5,003, 1.37%). Examination of the distribution of SSR motif types within a chromosome suggested the abundance of hexanucleotide repeats in each chromosome followed by dinucleotides, and these results are consistent with ‘Tunisia’ genome features as a whole. Concerning major repeat types, AT/AG was the most frequent (14.16%), followed by AAAAAT/AAAAAG (7.89%), A/C (7.54%), AAT/AAG (5.23%), AAAT/AAAG (4.37%), and AAAAT/AAAAG (1.2%) types. We designed and validated a total of 3,839 class I SSRs in the ‘Tunisia’ genome through electronic polymerase chain reaction (ePCR) and found 1,165 (30.34%) SSRs producing a single amplicon. Then, we selected 906 highly variable SSRs (> 40 nt) from the ePCR-verified class I SSRs and in silico validated across multiple draft genomes of pomegranate, which provided us a subset of 265 highly polymorphic SSRs. Of these, 235 primers were validated on six pomegranate genotypes through wet-lab experiment. We found 221 (94%) polymorphic SSRs on six genotypes, and 187 of these SSRs had ≥ 0.5 PIC values. The utility of the developed SSRs was demonstrated by analyzing genetic diversity of 30 pomegranate genotypes using 16 HvSSRs spanning eight pomegranate chromosomes. In summary, we developed a comprehensive set of highly polymorphic genome-wide SSRs. These chromosome-specific SSRs will serve as a powerful genomic tool to leverage future genetic studies, germplasm management, and genomics-assisted breeding in pomegranate.

Comparison of the Microsatellite Distribution Patterns in the Genomes of Euarchontoglires at the Taxonomic Level.

Microsatellite or simple sequence repeat (SSR) instability within genes can induce genetic variation. The SSR signatures remain largely unknown in different clades within Euarchontoglires, one of the most successful mammalian radiations. Here, we conducted a genome-wide characterization of microsatellite distribution patterns at different taxonomic levels in 153 Euarchontoglires genomes. Our results showed that the abundance and density of the SSRs were significantly positively correlated with primate genome size, but no significant relationship with the genome size of rodents was found. Furthermore, a higher level of complexity for perfect SSR (P-SSR) attributes was observed in rodents than in primates. The most frequent type of P-SSR was the mononucleotide P-SSR in the genomes of primates, tree shrews, and colugos, while mononucleotide or dinucleotide motif types were dominant in the genomes of rodents and lagomorphs. Furthermore, (A)n was the most abundant motif in primate genomes, but (A)n, (AC)n, or (AG)n was the most abundant motif in rodent genomes which even varied within the same genus. The GC content and the repeat copy numbers of P-SSRs varied in different species when compared at different taxonomic levels, reflecting underlying differences in SSR mutation processes. Notably, the CDSs containing P-SSRs were categorized by functions and pathways using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes annotations, highlighting their roles in transcription regulation. Generally, this work will aid future studies of the functional roles of the taxonomic features of microsatellites during the evolution of mammals in Euarchontoglires.

Development and application of perfect SSR markers in cotton

BackgroundThis study aimed to develop a set of perfect simple sequence repeat (SSR) markers with a single copy in the cotton genome, to construct a DNA fingerprint database suitable for authentication of cotton cultivars. We optimized the polymerase chain reaction (PCR) system for multi-platform compatibility and improving detection efficiency. Based on the reference genome of upland cotton and 10× resequencing data of 48 basic cotton germplasm lines, single-copy polymorphic SSR sites were identified and developed as diploidization SSR markers. The SSR markers were detected by denaturing polyacrylamide gel electrophoresis (PAGE) for initial screening, then fluorescence capillary electrophoresis for secondary screening. The final perfect SSR markers were evaluated and verified using 210 lines from different sources among Chinese cotton regional trials.ResultsUsing bioinformatics techniques, 1 246 SSR markers were designed from 26 626 single-copy SSR loci. Adopting a stepwise (primary and secondary) screening strategy, a set of 60 perfect SSR markers was selected with high amplification efficiency and stability, easy interpretation of peak type, multiple allelic variations, high polymorphism information content (PIC) value, uniform chromosome distribution, and single-copy characteristics. A multiplex PCR system was established with ten SSR markers using capillary electrophoresis detection.ConclusionsA set of perfect SSR markers of cotton was developed and a high-throughput SSR marker detection system was established. This study lays a foundation for large-scale and standardized construction of a cotton DNA fingerprint database for authentication of cotton varieties.

SSRMMD: A Rapid and Accurate Algorithm for Mining SSR Feature Loci and Candidate Polymorphic SSRs Based on Assembled Sequences.

Microsatellites or simple sequence repeats (SSRs) are short tandem repeats of DNA widespread in genomes and transcriptomes of diverse organisms and are used in various genetic studies. Few software programs that mine SSRs can be further used to mine polymorphic SSRs, and these programs have poor portability, have slow computational speed, are highly dependent on other programs, and have low marker development rates. In this study, we develop an algorithm named Simple Sequence Repeat Molecular Marker Developer (SSRMMD), which uses improved regular expressions to rapidly and exhaustively mine perfect SSR loci from any size of assembled sequence. To mine polymorphic SSRs, SSRMMD uses a novel three-stage method to assess the conservativeness of SSR flanking sequences and then uses the sliding window method to fragment each assembled sequence to assess its uniqueness. Furthermore, molecular biology assays support the polymorphic SSRs identified by SSRMMD. SSRMMD is implemented using the Perl programming language and can be downloaded from https://github.com/GouXiangJian/SSRMMD.

Comparison of microsatellite distribution patterns in twenty-nine beetle genomes.

Simple sequence repeats (SSRs) represent an important source of genetic variation that provides a basis for adaptation to different environments in organisms. In this study, we examined the distribution patterns of SSRs in twenty-nine beetle genomes and carried out Gene Ontology (GO) analysis of CDSs embedded with perfect SSRs (P-SSRs). The results demonstrated that imperfect SSRs (I-SSRs) represented the most abundant SSR category in beetle genomes and in different genomic regions (CDS, exon, and intron regions). The numbers of P-SSRs, I-SSRs, compound SSRs, and variable number tandem repeats were positively correlated with beetle genome size, whereas neither the frequency nor the density of the SSRs was correlated with genome size. Moreover, our results demonstrated that common genomic features of P-SSRs within the same suborder or family of Coleoptera were rare. Mono-, di-, tri-, or tetranucleotide SSRs were the most abundant P-SSR categories in beetle genomes. The preferred predominant repeat motif among the mononucleotide P-SSRs was (A)n, but the most frequent repeat motifs for other length classes varied differentially among these genomes. Furthermore, the P-SSR type with the highest GC content differed in the beetle genomes and in different genomic regions. CV (coefficient of variability) analysis demonstrated that the repeat copy numbers of P-SSRs presented relatively higher variation in introns than in CDSs and exons. The GO terms of CDSs containing P-SSRs for molecular functions were mainly enriched in "binding" and "transcription". Our findings will be useful for studying the functional roles of microsatellite heterogeneity in beetle adaptation.