In-silico analysis of simple and imperfect microsatellites in diverse tobamovirus genomes

An in-silico analysis of simple sequence repeats (SSRs) in 30 species of tobamoviruses was done. SSRs (mono to hexa) were present with variant frequency across species. Compound microsatellites, primarily of variant motifs accounted for up to 11.43% of the SSRs. Motif duplications were observed for A, T, AT, and ACA repeats. (AG)–(TC) was the most prevalent SSR-couple. SSRs were differentially localized in the coding region with ~ 54% on the 128 kDa protein while 20.37% was exclusive to 186 kDa protein. Characterization of such variations is important for elucidating the origin, sequence variations, and structure of these widely used, but incompletely understood sequences.     

蜜蜂EST中的微卫星分析

为加速分子标记在蜜蜂遗传、进化与行为等方面的利用,分析了简单重复序列(Simple Sequence Repeats,SSRs)在蜜蜂EST中的分布频率与密度。所分析的蜜蜂EST数据集包含15869条序列,总长为7．9Mb。结果显示,蜜蜂ESTs中SSRs的频率为1／0．52kb,其中6碱基重复基序占总SSRs的45．0％,是最丰富的重复单元,而2、1、3、4与5碱基重复基序分别占总SSRs的17．9％、14．1％、11．6％、9．2％和2．2％。同时,在各种SSRs重复单元中,富含A碱基的重复单元占据优势地位,如：A、AT、AG、AC、AAT、AAG、AAC、AAAT、AAAG、AAAAG、AAAAT、AATAT、AAAAAG和AAAAAT重复基序,而富含G碱基的重复单元在基因编码区中含量较低。进一步分析显示：蜜蜂SSRs在冗余与非冗余EST数据集中的分布频率与密度相似,仅存在极小的偏差,表明可从现有的部分ESTs数据中方便地获取有效的微卫星标记。     

Motif mismatches in microsatellites: insights from genome-wide investigation among 20 insect species

We present a detailed genome-wide comparative study of motif mismatches of microsatellites among 20 insect species representing five taxonomic orders. The results show that varying proportions (∼15–46%) of microsatellites identified in these species are imperfect in motif structure, and that they also vary in chromosomal distribution within genomes. It was observed that the genomic abundance of imperfect repeats is significantly associated with the length and number of motif mismatches of microsatellites. Furthermore, microsatellites with a higher number of mismatches tend to have lower abundance in the genome, suggesting that sequence heterogeneity of repeat motifs is a key determinant of genomic abundance of microsatellites. This relationship seems to be a general feature of microsatellites even in unrelated species such as yeast, roundworm, mouse and human. We provide a mechanistic explanation of the evolutionary link between motif heterogeneity and genomic abundance of microsatellites by examining the patterns of motif mismatches and allele sequences of single-nucleotide polymorphisms identified within microsatellite loci. Using Drosophila Reference Genetic Panel data, we further show that pattern of allelic variation modulates motif heterogeneity of microsatellites, and provide estimates of allele age of specific imperfect microsatellites found within protein-coding genes.     

Incidence,complexity and diversity of simple sequence repeats across potexvirus genomes

An in-silico analysis of simple sequence repeats (SSRs) in genomes of 32 species of potexviruses was performed wherein a total of 691 SSRs and 33 cSSRs were observed. Though SSRs were present in all the studied genomes their incident frequency ranged from 11 to 30 per genome. Further, 10 potexvirus genomes possessed no cSSRs when extracted at a dMAX of 10 and wherein present, the highest frequency was 3. SSR and cSSR incidence, relative density and relative abundance were non-significantly correlated with genome size and GC content suggesting an ongoing evolutionary and adaptive phase of the virus species. SSRs present primarily ranged from mono- to tri-nucleotide repeat motifs with a greatly skewed distribution across the coding and non-coding regions. Present work is an effort for the undergoing compilation and analysis of incidence, distribution and variation of the viral repeat sequences to understand their evolutionary and functional relevance.     

核盘菌和灰葡萄孢基因组中的简单重复序列分析

利用公共的真菌基因组数据库资源, 对核盘菌（Sclerotinia sclerotiorum）和灰葡萄孢（Botrytis cinerea）基因组中SSRs的结构类型、分布、丰度及最长序列等进行了系统分析, 并与已经研究过的禾谷镰孢菌（Fusarium graminearum）, 稻瘟病菌（Magnaporthe grisea）和黑粉菌（Ustilago maydis）等几种植物病原真菌基因组中的SSRs进行了比较。结果表明: 核盘菌和灰葡萄孢基因组中的SSRs非常丰富, 分别为6 539和8 627个, 并且在结构类型和分布规律上具有一定的相似性; 与其他几种病原真菌相比, 核盘菌和灰葡萄孢基因组中长重复的四、五、六核苷酸基序更为丰富, 从而使得这两种真菌具有更高的变异性。同时, 我们发现真菌基因组中SSRs的丰度与基因组的大小及GC含量没有必然的关系。文章对核盘菌和灰葡萄孢基因组中SSRs的丰度、出现频率及最长基序的分析为快速、便捷地设计多态性丰富的SSRs引物提供了有益的信息。     

Survey of simple sequence repeats in completed fungal genomes

The use of simple sequence repeats or microsatellites as genetic markers has become very popular because of their abundance and length variation between different individuals. SSRs are tandem repeat units of 1 to 6 base pairs that are found abundantly in many prokaryotic and eukaryotic genomes. This is the first study examining and comparing SSRs in completely sequenced fungal genomes. We analyzed and compared the occurrences, relative abundance, relative density, most common, and longest SSRs in nine taxonomically different fungal species: Aspergillus nidulans, Cryptococcus neoformans, Encephalitozoon cuniculi, Fusarium graminearum, Magnaporthe grisea, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Ustilago maydis. Our analysis revealed that, in all of the genomes studied, the occurrence, abundance, and relative density of SSRs varied and was not influenced by the genome sizes. No correlation between relative abundance and the genome sizes was observed, but it was shown that N. crassa, the largest genome analyzed had the highest relative abundance of SSRs. In most genomes, mononucleotide, dinucleotide, and trinucleotide repeats were more abundant than the longer repeated SSRs. Generally, in each organism, the occurrence, relative abundance, and relative density of SSRs decreased as the repeat unit increased. Furthermore, each organism had its own common and longest SSRs. Our analysis showed that the relative abundance of SSRs in fungi is low compared with the human genome and that longer SSRs in fungi are rare. In addition to providing new information concerning the abundance of SSRs for each of these fungi, the results provide a general source of molecular markers that could be useful for a variety of applications such as population genetics and strain identification of fungal organisms.     

Distribution and evolution of short tandem repeats in closely related bacterial genomes

Simultaneous identification and comparison of perfect and imperfect microsatellites within a genome is a valuable tool both to overcome the lack of a consensus definition of SSRs and to assess repeat history. Detailed analysis of the overall distribution of perfect and imperfect microsatellites in closely related bacterial taxa is expected to give new insight into the evolution of prokaryotic genomes. We have performed a genome-wide analysis of microsatellite distribution in four Escherichia coli and seven Chlamydial strains. Chlamydial strains generally have a higher density of SSRs and show greater intra-group differences of SSR distribution patterns than E. coli genomes. In most investigated genomes the distribution of the total lengths of matching perfect and imperfect trinucleotide repeats are highly similar, with the notable exception of C. muridarum. Closely related strains show more similar repeat distribution patterns than strains separated by a longer divergence time. The discrepancy between the preferred classes of perfect and imperfect repeats in C. muridarum implies accelerated evolution of SSRs in this particular strain. Our results suggest that microsatellites, although considerably less abundant than in eukaryotic genomes, may nevertheless play an important role in the evolution of prokaryotic genomes and several gene families.     

The analysis of microsatellites and compound microsatellites in 56 complete genomes of Herpesvirales

Simple sequence repeats (SSRs), or microsatellites, are special DNA/RNA sequences with repeated unit of 1–6 bp. The genomes of Herpesvirales have many repeating structures, which is an excellent system to study the evolution and roles of microsatellites and compound microsatellites in viruses. Therefore, 56 genomes of Herpesvirales were selected and the occurrence, composition and complexity of different repeats were investigated in the genomes. A total of 63,939 microsatellites and 5825 compound microsatellites were extracted from 56 genomes. It found that GC content has a significant strong correlation with both the counts of microsatellites (CM) and the counts of compound microsatellites (CCM). However, genome size has a moderate correlation only with CM and almost no correlation with CCM. The compound microsatellites occurring in genic regions are obviously more than that in intergenic regions. In general, the number of compound microsatellite decreases with the increase of complexity (C) (the count of individual microsatellites being part of a compound microsatellite) and the complexity hardly exceeds C = 4. The vast majority of compound microsatellites exist in intergenic regions, when C ≥ 10. The distributions of SSRs tend to be organism-specific rather than host-specific in herpesvirus genomes. The diversity of microsatellites and compound microsatellites may be helpful for a better understanding of the viral genetic diversity, genotyping, and evolutionary biology in herpesviruses genomes.     

Optimized construction of microsatellite-enriched libraries

The construction of microsatellite-enriched libraries is an indispensable tool to search for molecular markers as complete genome sequences are still not available for the majority of species of interest. Numerous protocols are available in the literature for the construction of these libraries; however, sometimes their low efficiency or lack of optimization in the protocols can restrict their efficacy. We have designed and tested various adapters and ligation methods; we also tested oligo-repeat combinations and hybridization temperatures, and created libraries with this new protocol for four organisms: Ipomoea batatas (L.) Lam, Chionanthus retusus Lindley & Paxton, Rotylenchulus reniformis Linford & Olivera and Puccinia kuehnii W. Krüger. The number of microsatellites detected for these species ranged from 2494 to 3919 per Mb of nonredundant sequence, that was 0.86 and 1.53 microsatellites per contig, with 37-66% of di-nucleotide motifs and 21-49% of tri- to octa-nucleotide repeats combined. A simplified protocol is provided for the successful generation of SSR-enriched libraries.     

Compound microsatellites in complete Escherichia coli genomes

Compound microsatellites consisting of two or more repeats in close proximity have been found in eukaryotic genomes. So far such compound microsatellites have not been investigated in any prokaryotic genomes. We have therefore examined compound microsatellites in 22 complete genomes of Escherichia coli, which is one of the ideal model organisms to analyze the nature and evolution of prokaryotic compound microsatellites. Our results indicated that about 1.75-2.85% of all microsatellites could be accounted as compound microsatellites with very low complexity, and most compound microsatellites were composed of very different motifs. Compound microsatellites were significantly overrepresented in all surveyed genomes. These results were dramatically different from those in eukaryotes. We discussed the possible reasons for the observed divergence.     

Low microsatellite frequencies in neuron and brain expressed microRNAs

The locations of microsatellites in mammalian genomes are restricted by purifying selection in a number of ways. For example, with the exception of some trinucleotide repeats they are excluded from protein coding regions of genomes because of their tendency to cause frameshift mutations. Here we investigate whether purifying selection might affect the types and frequencies of microsatellites in microRNA (miRNA). We concentrate on miRNAs expressed in neurons and the brain (NB-miRNAs) as microsatellites in these genes might give rise to similar effects as disease-causing repeats in protein coding genes. We show that in human miRNAs in general AG and AT microsatellites are reduced in frequency compared to AC repeats and that NB-miRNA genes contain significantly fewer microsatellites than expected from frequencies of microsatellites in other miRNA genes. NB-miRNAs show lower levels of sequence divergence in comparisons of human-macaque orthologues and more often have detectable orthologues in non-human mammals than non-NB-miRNAs. This suggests that microsatellites in miRNAs may indeed be constrained by purifying selection and that the strength of this selection may differ between NB-miRNAs and non-NB-miRNAs. We identify a number of ways in which the potential disruption of pre-miRNA secondary structure might result in purifying selection. However other, non-selective forces could also play a role in generating the biases observed in miRNA microsatellites.     

Development and Characterization of Simple Sequence Repeat Markers Providing Genome-Wide Coverage and High Resolution in Maize

Simple sequence repeats (SSRs) have been widely used in maize genetics and breeding, because they are co-dominant, easy to score, and highly abundant. In this study, we used whole-genome sequences from 16 maize inbreds and 1 wild relative to determine SSR abundance and to develop a set of high-density polymorphic SSR markers. A total of 264 658 SSRs were identified across the 17 genomes, with an average of 135 693 SSRs per genome. Marker density was one SSR every of 15.48 kb. (C/G)n, (AT)n, (CAG/CTG)n, and (AAAT/ATTT)n were the most frequent motifs for mono, di-, tri-, and tetra-nucleotide SSRs, respectively. SSRs were most abundant in intergenic region and least frequent in untranslated regions, as revealed by comparing SSR distributions of three representative resequenced genomes. Comparing SSR sequences and e-polymerase chain reaction analysis among the 17 tested genomes created a new database, including 111 887 SSRs, that could be develop as polymorphic markers in silico. Among these markers, 58.00, 26.09, 7.20, 3.00, 3.93, and 1.78% of them had mono, di-, tri-, tetra-, penta-, and hexa-nucleotide motifs, respectively. Polymorphic information content for 35 573 polymorphic SSRs out of 111 887 loci varied from 0.05 to 0.83, with an average of 0.31 in the 17 tested genomes. Experimental validation of polymorphic SSR markers showed that over 70% of the primer pairs could generate the target bands with length polymorphism, and these markers would be very powerful when they are used for genetic populations derived from various types of maize germplasms that were sampled for this study.     

Genome-wide analysis of simple sequence repeats in the model medicinal mushroom Ganoderma lucidum

Simple sequence repeats (SSRs) or microsatellites are one of the most popular sources of genetic markers and play a significant role in gene function and genome organization. We identified SSRs in the genome of Ganoderma lucidum and analyzed their frequency and distribution in different genomic regions. We also compared the SSRs in G. lucidum with six other Agaricomycetes genomes: Coprinopsis cinerea, Laccaria bicolor, Phanerochaete chrysosporium, Postia placenta, Schizophyllum commune and Serpula lacrymans. Based on our search criteria, the total number of SSRs found ranged from 1206 to 6104 and covered from 0.04% to 0.15% of the fungal genomes. The SSR abundance was not correlated with the genome size, and mono- to tri-nucleotide repeats outnumbered other SSR categories in all of the species examined. In G. lucidum, a repertoire of 2674 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. The highest SSR relative abundance was found in introns (108 SSRs/Mb), followed by intergenic regions (84 SSRs/Mb). A total of 684 SSRs were found in the protein-coding sequences (CDSs) of 588 gene models, with 81.4% of them being tri- or hexa-nucleotides. After scanning for InterPro domains, 280 of these genes were successfully annotated, and 215 of them could be assigned to Gene Ontology (GO) terms. SSRs were also identified in 28 bioactive compound synthesis-related gene models, including one 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), three polysaccharide biosynthesis genes and 24 cytochrome P450 monooxygenases (CYPs). Primers were designed for the identified SSR loci, providing the basis for the future development of SSR markers of this medicinal fungus.     

Genome-Wide Comparative Analyses of Microsatellites in Papaya

Microsatellites, or simple sequence repeats (SSRs), are highly polymorphic and universally distributed in eukaryotes. SSRs have been used extensively as sequence tagged markers in genetic studies. Recently, the functional and evolutionary importance of SSRs has received considerable attention. Here we report the mining and characterization of the SSRs in papaya genome. We analyzed SSRs from 277.4 Mb of whole genome shotgun (WGS) sequences, 51.2 Mb bacterial artificial chromosome (BAC) end sequences (BES), and 13.4 Mb expressed sequence tag (EST) sequences. The papaya SSR density was one SSR per 0.7 kb of DNA sequence in the WGS, which was higher than that in BES and EST sequences. SSR abundance was dramatically reduced as the repeat length increased. According to SSR motif length, dinucleotide repeats were the most common motif in class I, whereas hexanucleotides were the most copious in class II SSRs. The tri- and hexanucleotide repeats of both classes were greater in EST sequences compared to genomic sequences. In class I SSR, AT and AAT were the most frequent motifs in BES and WGS sequences. By contrast, AG and AAG were the most abundant in EST sequences. For SSR marker development, 9,860 primer pairs were surveyed for amplification and polymorphism. Successful amplification and polymorphic rates were 66.6% and 17.6%, respectively. The highest polymorphic rates were achieved by AT, AG, and ATG motifs. The genome wide analysis of microsatellites revealed their frequency and distribution in papaya genome, which varies among plant genomes. This complete set of SSRs markers throughout the genome will assist diverse genetic studies in papaya and related species.     

Consensus features of microsatellite distribution: Microsatellite contents are universally correlated with recombination rates and are preferentially depressed by centromeres in multicellular eukaryotic genomes

Microsatellite DNA is highly polymorphic and informative, which makes its distribution pattern and its associations very valuable for marker applications and genomic research in evolution. Using computational and statistical approaches, based on database technology, we have demonstrated that microsatellite content is consistently and significantly 2 to 5 fold lower than the average chromosomal level in the centromeric and pericentromeric regions of the chromosomes of two plant species, Arabidopsis thaliana and Oryza sativa. We conducted a path coefficient analysis to compare the direct effect of microsatellites (from mono-nucleotide through to penta-nucleotide repeats) on recombination rates. The results revealed that tri- and penta-nucleotide microsatellites significantly influence recombination rates. In the human genome, tri-, tetra- and mono-nucleotide microsatellites, in decreasing order, make significant direct contributions to recombination rates, according to DECODE, GENTHON, and MARSHFIELD averages. Path coefficient analysis in rice and human genomes of the impact of di-nucleotide microsatellites of different motifs on recombination rates indicate that motifs with either A or T have an effect, resulting in increased recombination rates for microsatellites with motifs consisting of 50% A or T, such as AG, TC, CA, TG. Conversely, microsatellites with motifs consisting of only A & T or G & C, such as AT, TA, GC or CG, have decreased recombination rates. The extremely low microsatellite content in centromeric and pericentromeric regions, as well as the quantitative association of microsatellite sequences with the recombination rate at the genome level, suggests that purifying selection in genome evolution creates a balance between genomic polymorphisms and the biological function of sequences in a genome.     

Chloroplast microsatellites in Prunus,Rosaceae

Ten chloroplast microsatellite markers were developed from Japanese plum (Prunus salicina) based on nucleotide sequences of c. 4300 bp from six chloroplast regions. Out of 10 microsatellites, seven markers contained mononucleotide repeats. Almost all microsatellites displayed discrete amplified fragments for 17 species in Prunus. The microsatellites generated 46 different fragment types and differentiated all used species. Polymorphism was also observed within species for all microsatellites.     

Barley microsatellites: allele variation and mapping

Microsatellites have developed into a powerful tool for mapping mammalian genomes and first reports about their use in plants have been published. A database search of 228 barley sequences from GenBank and EMBL was made to determine which simple sequence repeat (SSR) motif prevails in barley. Nearly all types of SSRs were found. The (A)n and (T)n SSRs occurred more often than (C)n and (G)n for n10. Among the dinucleotide repeats, the (CG)n SSRs occurred least often. Trinucleotide repeats did not occur with n>7 and there is no correlation between the GC content in the trinucleotide motifs and the number of observed SSRs. Analysing 15 different microsatellites with 11 barleys yielded 2.1 alleles per microsatellite. Sequencing 25 putative microsatellites showed that the resolution capacity of highquality agarose gels was sufficient to determine differences of only three base paris. Five microsatellites were mapped on three different chromosomes of a barley RFLP map.     

Characterization and cross‐species amplification of microsatellite markers in cherimoya (Annona cherimola Mill., Annonaceae)

Fifteen polymorphic microsatellites were developed using a CT/AG‐enriched genomic library of cherimoya cv. Fino de Jete and screened on 23 genotypes from different geographical areas. This is the first set of microsatellites developed in Annonaceae. Fourteen of the microsatellites detected a single locus and only one detected two loci. The number of alleles in the single locus microsatellites ranged from two to six and the observed and expected heterozygosity ranged from 0.17 to 0.61 and from 0.12 to 0.76, respectively. Most of the simple sequence repeats were transferable to other species in the family.     

Distribution and characterization of simple sequence repeats in Gossypium raimondii genome

Simple sequence repeats (SSRs) can be derived from the complete genome sequence. These markers are important for gene mapping as well as marker-assisted selection (MAS). To develop SSRs for cotton gene mapping, we selected the complete genome sequence of Gossypium raimondii, which consisted of 4447 non-redundant scaffolds. Out of 775.2 Mb sequence examined, a total of 136,345 microsatellites were identified with a density of 5.69 kb per SSR in the G. raimondii genome leading to development of 112,177 primer pairs. The distributions of SSRs in the genome were non-random. Among the different motifs ranging from 1 to 6 bp, penta-nucleotide repeats were most abundant (30.5%), followed by tetra-nucleotide repeats (18.2%) and di-nucleotide repeats (16.9%). Among all identified 457 motif types, the most frequently occurring repeat motifs were poly-AT/TA, which accounted for 79.8% of the total di-nt SSRs, followed by AAAT/TTTA with 51.5% of the total tetra-nucleotede. Further, 18,834 microsatellites were detected from the protein-coding genes, and the frequency of gene containing SSRs was 46.0% in 40,976 genes of G. raimondii. These genome-based SSRs developed in the present study will lay the groundwork for developing large numbers of SSR markers for genetic mapping, gene discovery, genetic diversity analysis, and MAS breeding in cotton.