Creation of a SINE enriched library for the isolation of polymorphic (AGC)n microsatellite markers in the bovine genome

Trinucleotide (AGC)_n microsatellites are found as 3′ tails of the artiodactyl short interspersed nuclear element (SINE) A-dimer. We describe a polymerase chain reaction (PCR)-based method for the construction of a plasmid library enriched for SINE (AGC)_n microsatellites. By amplifying Sau3AI inserts with a conserved SINE primer and a flanking vector primer, a 35-fold enrichment of (AGC)_n microsatellites over a conventional genomic library was obtained. The SINE primer was used for both sequencing of AGC-containing inserts and analysis of polymorphism. Twenty-three unique reverse primers were synthesized and used on bovine genomic DNA, 21 producing PCR products of expected size. Five polymorphic (AGC)_n microsatellites with 2–4 alleles each were characterized. Allele sizes differed by a 3 bp motif and lacked the stutter bands associated with dinucleotide repeats. A tendency of increased polymorphism for longer AGC repeat arrays was observed. High stringency selection for positive clones containing eight or more AGC repeats can thus facilitate the isolation of polymorphic (AGC)_n microsatellites, Enrichment for (AGC), microsatellites by SINE-vector PCR can be applied to other bovidae species, such as sheep or goat, containing the artiodactyl SINE elements.     

Characterization of rabbit DNA micros extracted from the EMBL nucleotide sequence database

Microsatellite polymorphisms are invaluable for mapping vertebrate genomes. In order to estimate the occurrence of microsatellites in the rabbit genome and to assess their feasibility as markers in rabbit genetics, a survey on the presence of all types of mononucleotide, dinucleotide, trinucleotide and tetranucleotide repeats, with a length of about 20 bp or more, was conducted by searching the published rabbit DNA sequences in the EMBL nucleotide database (version 32). A total of 181 rabbit microsatellites could be extracted from the present database. The estimated frequency of microsatellites in the rabbit genome was one microsatellite for every 2–3 kb of DNA. Dinucleotide repeats constituted the prevailing class of microsatellites, followed by trinucleotide, mononucleotide and tetranucleotide repeats, respectively. The average length of the microsatellites, as found in the database, was 26, 23, 23 and 22 bp for mono-, di-, tri- and tetranucleotide repeats, respectively. The most common repeat motif was AG, followed by A, AC, AGG and CCG. This group comprised about 70% of all extracted rabbit microsatellites. About 61% of the microsatellites were found in non-coding regions of genes, whereas 15% resided in (protein) coding regions. A significant fraction of rabbit microsatellites (about 22%) was found within interspersed repetitive DNA sequences.     

Fosmid library construction and end sequences analysis of the Pacific oyster,Crassostrea gigas

The Pacific oyster (Crassostrea gigas) is globally distributed and is one of the most commercially and ecologically important marine organisms. However, little is known about the genome of this species. In this study, a C. gigas fosmid library was constructed that contains 459,936 clones with an average insert size of approximately 40 kb, representing 22.34-fold haploid genome equivalents. End sequencing generated 90,240 fosmid end sequences (FESs) with an average length of 384.27 base pairs (bp), covering approximately 2.58% of the Pacific oyster genome. The FESs were subsequently assembled and annotated, resulting in 6332 sequences with predicted open reading frames≥300 and 1,189,100 bp repeats. Furthermore, a total of 3200 microsatellite repeats were identified, and dinucleotide repeats were found to occur most abundantly, with AG and AAT being the most abundant repeat class of dinucleotides and trinucleotides. We also found that the repeat number was generally negatively proportional to the repeat element length. Microsatellites composition between the transcribed sequences and genomic sequences was shown to be different. Point mutations of microsatellite were non-random and underwent strong selection stress. Overall, a comprehensive sequence resource for the Pacific oyster was created, including annotated transposable elements, tandem repeats, protein coding sequences and microsatellites. These initial findings will serve as resources for further in-depth studies of physical mapping, gene discovery, microsatellite marker developing and evolution studies.     

A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene.

The human genome contains approximately 50,000 copies of an interspersed repeat with the sequence (dT-dG)n, where n = approximately 10-60. In humans, (TG)n repeats have been found in several sequenced regions. Since minisatellite regions with larger repeat elements often display extensive length polymorphisms, we suspected that (TG)n repeats ("microsatellites") might also be polymorphic. Using the polymerase chain reaction to amplify a (TG)n microsatellite in the human cardiac actin gene, we detected 12 different allelic fragments in 37 unrelated individuals, 32 of whom were heterozygous. Codominant Mendelian inheritance of fragments was observed in three families with a total of 24 children. Because of the widespread distribution of (TG)n microsatellites, polymorphisms of this type may be generally abundant and present in regions where minisatellites are rare, making such microsatellite loci very useful for linkage studies in humans.     

Survey and analysis of microsatellites in the silkworm, Bombyx mori: frequency, distribution, mutations, marker potential and their conservation in heterologous species

We studied microsatellite frequency and distribution in 21.76-Mb random genomic sequences, 0.67-Mb BAC sequences from the Z chromosome, and 6.3-Mb EST sequences of Bombyx mori. We mined microsatellites of >/=15 bases of mononucleotide repeats and >/=5 repeat units of other classes of repeats. We estimated that microsatellites account for 0.31% of the genome of B. mori. Microsatellite tracts of A, AT, and ATT were the most abundant whereas their number drastically decreased as the length of the repeat motif increased. In general, tri- and hexanucleotide repeats were overrepresented in the transcribed sequences except TAA, GTA, and TGA, which were in excess in genomic sequences. The Z chromosome sequences contained shorter repeat types than the rest of the chromosomes in addition to a higher abundance of AT-rich repeats. Our results showed that base composition of the flanking sequence has an influence on the origin and evolution of microsatellites. Transitions/transversions were high in microsatellites of ESTs, whereas the genomic sequence had an equal number of substitutions and indels. The average heterozygosity value for 23 polymorphic microsatellite loci surveyed in 13 diverse silkmoth strains having 2-14 alleles was 0.54. Only 36 (18.2%) of 198 microsatellite loci were polymorphic between the two divergent silkworm populations and 10 (5%) loci revealed null alleles. The microsatellite map generated using these polymorphic markers resulted in 8 linkage groups. B. mori microsatellite loci were the most conserved in its immediate ancestor, B. mandarina, followed by the wild saturniid silkmoth, Antheraea assama.     

Map and analysis of microsatellites in the genome of <Emphasis Type="Italic">Populus</Emphasis>: The first sequenced perennial plant

We mapped and analyzed the microsatellites throughout 284295605 base pairs of the unambiguously assembled sequence scaffolds along 19 chromosomes of the haploid poplar genome. Totally, we found 150985 SSRs with repeat unit lengths between 2 and 5 bp. The established microsatellite physical map demonstrated that SSRs were distributed relatively evenly across the genome of Populus. On average, These SSRs occurred every 1883 bp within the poplar genome and the SSR densities in intergenic regions, introns, exons and UTRs were 85.4%, 10.7%, 2.7% and 1.2%, respectively. We took di-, tri-, tetra-and pentamers as the four classes of repeat units and found that the density of each class of SSRs decreased with the repeat unit lengths except for the tetranucleotide repeats. It was noteworthy that the length diversification of microsatellite sequences was negatively correlated with their repeat unit length and the SSRs with shorter repeat units gained repeats faster than the SSRs with longer repeat units. We also found that the GC content of poplar sequence significantly correlated with densities of SSRs with uneven repeat unit lengths (tri-and penta-), but had no significant correlation with densities of SSRs with even repeat unit lengths (di-and tetra-). In poplar genome, there were evidences that the occurrence of different microsatellites was under selection and the GC content in SSR sequences was found to significantly relate to the functional importance of microsatellites.     

桉树EST序列中微卫星含量及相关特征

通过对桉树属(Eucalyptus)的10 000条EST序列进行分析, 在其中的1 499条序列上共发现1 775个微卫星重复序列。含有微卫星的EST序列约占序列总数的15%。此外, 还发现桉树EST序列所含微卫星长度的变异速率与重复单元长度呈负相关; 微卫星的丰度与重复单元长度也呈负相关(三碱基重复微卫星除外)。在桉树EST序列中, 重复单元长度为三碱基的微卫星最为丰富。三碱基重复单元微卫星的过度富集可能是由于遗传密码选择所致。在微卫星的丰度及长度变异方面, 桉树EST序列与杨树(Populus trichocarpa)基因组注释的转录序列随重复单元长度的变化呈现出相同的规律, 但桉树EST序列中微卫星频率及三碱基重复微卫星的含量显著偏低, 推测含微卫星的基因表达丰度极有可能低于不含微卫星的基因。通过对发现的所有微卫星位点进行引物设计, 并对设计的引物进行PCR检测, 结果表明所设计的引物具有极高的扩增成功率。     

Map and analysis of microsatellites in the genome of Populus: The first sequenced perennial plant

Environmental Sciences Division, Oak Ridge National Laboratory, TN, USA We mapped and analyzed the microsatellites throughout 284295605 base pairs of the unambiguously assembled sequence scaffolds along 19 chromosomes of the haploid poplar genome. Totally, we found 150985 SSRs with repeat unit lengths between 2 and 5 bp. The established microsatellite physical map demonstrated tr at SSRs were distributed relatively evenly across the genome of Populus. On average, These SSRs occurred every 1883 bp within the poplar genome and the SSR densities in intergenic regions, introns, exons and UTRs were 85.4%, 10.7%, 2.7% and 1.2%, respectively. We took di-, tri-, tetra-and pentamers as the four classes of repeat units and found that the density of each class of SSRs decreased with the repeat unit lengths except for the tetranucleotide repeats. It was noteworthy that the length diversification of microsatellite sequences was negatively correlated with their repeat unit length and the SSRs with shorter repeat units gained repeats faster than the SSRs with longer repeat units. We also found that the GC content of poplar sequence significantly correlated with densities of SSRs with uneven repeat unit lengths (tri-and penta-), but had no significant correlation with densities of SSRs with even repeat unit lengths (di-and tetra-). In poplar genome, there were evidences that the occurrence of different microsatellites was under selection and the GC content in SSR sequences was found to significantly relate to the functional importance of microsatellites.     

Identification of abundant and informative microsatellites from shrimp (Penaeus monodon) genome

Microsatellites were isolated from P. monodon genomic libraries by direct sequencing of recombinant clones without probe screening. Forty-nine out of 83 clones sequenced contained 99 microsatellite arrays of three or more repeats. When five or more and ten or more repeats were considered, 28 and 14 microsatellites were detected, respectively. The 99 microsatellites were classified as perfect (75%), imperfect (6%), compound perfect (3%) and compound imperfect (16%). The abundance of di-, tri-, tetra- and hexanucleotide repeats were 67%, 20%, 9% and 3%, respectively. The dinucleotide repeats included 36 (CT)n, 31 (GT)n, 17(AT)n and 3 (CG)n. One octanucleotide repeat (ATTTATTC)5 was found within a large repeat sequence. Optimal annealing temperatures were determined for PCR using 11 primer sets encompassing 15 microsatellites. Ten primer sets provided successful amplifications with allele sizes generally ranging from 139 to 410 bp. All these primers amplified polymorphic loci with PIC values ranging from 0.63 to 0.96. Two primer sets amplified additional bands which can easily be distinguished from the bands of the main locus. Three out of 10 P. monodon microsatellites also amplified alleles in P. vannamei. The abundance and informative nature of P. monodon microsatellites and their potential for cross-species amplification make them useful for genetic studies.     

桉树EST序列中微卫星含量及相关特征

通过对桉树属（Eucalyptus）的10000条EST序列进行分析,在其中的1499条序列上共发现1775个微卫星重复序列。含有微卫星的EST序列约占序列总数的15%。此外,还发现桉树EST序列所含微卫星长度的变异速率与重复单元长度呈负相关;微卫星的丰度与重复单元长度也呈负相关（三碱基重复微卫星除外）。在桉树EST序列中,重复单元长度为三碱基的微卫星最为丰富。三碱基重复单元微卫星的过度富集可能是由于遗传密码选择所致。在微卫星的丰度及长度变异方面,桉树EST序列与杨树（Populus trichocarpa）基因组注释的转录序列随重复单元长度的变化呈现出相同的规律,但桉树EST序列中微卫星频率及三碱基重复微卫星的含量显著偏低,推测含微卫星的基因表达丰度极有可能低于不含微卫星的基因。通过对发现的所有微卫星位点进行引物设计,并对设计的引物进行PCR检测,结果表明所设计的引物具有极高的扩增成功率。     

Drosophila virilis has long and highly polymorphic microsatellites

Comparative genomics is a powerful approach to inference of the dynamics of genome evolution. Most information about the evolution of microsatellites in the genus Drosophila has been obtained from Drosophila melanogaster. For comparison, we collected microsatellite data for the distantly related species Drosophila virilis. Screening about 0.5 Mb of nonredundant genomic sequence from GenBank, we identified 239 dinucleotide microsatellites. On average, D. virilis dinucleotides were significantly longer than D. melanogaster microsatellites (7.69 repeats vs. 6.75 repeats). Similarly, direct cloning of microsatellites resulted in a higher mean repeat number in D. virilis than in D. melanogaster (12.7 repeats vs. 12.2 repeats). Characterization of 11 microsatellite loci mapping to division 40-49 on the fourth chromosome of D. virilis indicated that D. virilis microsatellites are more variable than those of D. melanogaster.     

Characterization of AT-rich microsatellites in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Polymorphism of microsatellite markers is often associated with the simple sequence repeat motif targeted. AT-rich microsatellites tend to be highly variable and this appears to be notable, especially in legume genomes. To analyze the value of AT-rich microsatellites for common bean (Phaseolus vulgaris L.), we developed a total of 85 new microsatellite markers, 74 of which targeted ATA or other AT-rich motif loci and 11 of which were made for GA, CA or CAC motif loci. We evaluated the loci for the level of allelic diversity in comparison to previously characterized microsatellites using a panel of 18 standard genotypes and genetically mapped any loci polymorphic in the DOR364 × G19833 population. The majority of the microsatellites produced single bands and detected single loci, however, 15 of the AT-rich microsatellites produced multiple or double banding patterns; while only one of the GA or CA-rich microsatellites did. The polymorphism information content (PIC) values averaged 0.892 and 0.600 for the AT and ATA motif microsatellites, respectively, but only 0.140 for the CA-rich microsatellites. GA microsatellites, which had a large average number of repeats, had high to intermediate PIC, averaging 0.706. A total of 45 loci could be genetically mapped and distribution of the loci across the genome was skewed towards non-distal locations with a greater prevalence of loci on linkage groups b02, b09 and b11. AT-rich microsatellites were found to be a useful source of polymorphic markers for mapping and diversity assessment in common bean that appears to uncover higher diversity than other types of simple sequence repeat markers.     

Five polymorphic microsatellite VNTRs on the human X chromosome

The human genome contains approximately 50,000 copies of an interspersed repeat with the sequence (dT.dG/dA.dC)n, where n = approximately 10-60. We and others have found that several of these repeats have variable lengths in different individuals, with allelic fragments varying in size by multiples of 2 bp. These "microsatellite" variable number of tandem repeats (VNTRs) may be scored by PCR, using unique flanking primers to amplify the repeat-containing regions and resolving the products on DNA sequencing gels. Since few VNTRs have been found on the X chromosome, we screened a flow-sorted X chromosome-specific genomic library for microsatellites. Approximately 25% of the phage clones hybridized to a poly (dT-dG).poly(dA-dC) probe. Of seven X-linked microsatellites present in positive phages, five are polymorphic and three have both eight or more alleles and heterozygosities exceeding 75%. Using PCR to amplify genomic DNAs from hybrid cell panels, we confirmed the X localization of these VNTRs and regionally mapped four of them. The fifth VNTR was regionally mapped by virtue of its tight linkage to DXS87 in Centre du Polymorphisme Humain families. We conclude that whatever factors limit the occurrence of "classical" VNTRs and RFLPs on the X chromosome do not appear to operate in the case of microsatellite VNTRs.     

Microsatellite evolution: polarity of substitutions within repeats and neutrality of flanking sequences.

Though extensively used in a variety of disciplines, the evolutionary pattern of microsatellite sequences is still unclear. We addressed several questions relating to microsatellite evolution by analysing historically accumulated mutation events in a large set of artiodactyl (CA)n repeats, through sequence analysis of orthologous bovine and ovine loci. The substitution rate in microsatellite flanking sequences was not different from that in intron sequences, suggesting that if intron sequences in general are selectively neutral, sequences close to microsatellites are similarly so. This observation thus does not support the idea that successful heterologous amplification of microsatellites across distantly related taxa would be due to flanking sequences generally being under some form of selection. Interestingly, the substitution rate at the first nucleotide positions flanking repeats was significantly higher than in sequences further away. Moreover, the substitution rate in repeat units in the very end of microsatellites was significantly higher than that in the middle of repeat regions. Together these observations suggest a relative instability close to the boundary between repetitive and unique sequences. We present three models that potentially could explain such a feature, all involving inefficiency of mismatch repair systems.     

Microsatellite repeats are not randomly distributed within Norway spruce (Picea abies K.) expressed sequences.

A Norway spruce (Picea abies K.) cDNA library obtained from vegetative bud tissue was screened for the presence of (AG)n and (AC)n microsatellite repeats. Ten (AG)n and six (AC)n microsatellites were found, with an average length of 25.5 repeat units. Most of the microsatellites are simple perfect repeats. The microsatellite distribution within the clones is clearly non-random, with different classes of repeats lying in different positions relative to the coding region and in a highly conserved orientation. An estimate of the frequency of dinucleotide microsatellites in expressed regions was obtained, showing that SSRs (simple sequence repeats) are found in genes about 20 times less frequently than in random genomic clones, with (AG)n repeats more frequent than (AC)n repeats. Potential applications of these sequences as expressed region-based molecular markers are shown by developing six SSR markers for the detection of natural variation in Norway spruce populations and testing two of them for the identification of illegitimate progenies from a mapping population.     

In silico whole-genome EST analysis reveals 2322 novel microsatellites for the silver-lipped pearl oyster, Pinctada maxima

Molecular stock improvement techniques such as marker assisted selection have great potential in accelerating selective breeding programmes for animal production industries. However, the discovery and application of trait/marker associations usually requires a large number of genome-wide polymorphic loci. Here, we present 2322 unique microsatellites for the silver-lipped pearl oyster, Pinctada maxima, a species of aquaculture importance throughout the Indo-Australian Archipelago for production of the highly valued South Sea pearl. More than 1.2 million Roche 454 expressed sequence tag (EST) reads were screened for microsatellite repeat motifs. A total of 12,604 sequences contained either a di, tri, tetra, penta or hexa microsatellite repeat motif (n ≥ 6), with 6435 of these sequences having sufficient flanking regions for primer development. All identified microsatellites with designed primers were condensed into 2322 unique clusters (i.e., unique loci) of which 360 were shown to be polymorphic based on multiple sequence reads with different repeat motifs. Genotyping of five microsatellite loci demonstrated that in silico evaluation of polymorphism levels was a very useful method for identification of polymorphic loci, with the variation uncovered being a lower bound. Gene Ontology annotations of sequences containing microsatellites suggest that most are derived from a diverse array of unique genes. This EST derived microsatellite database will be a valuable resource for future studies in genetic map construction, diversity analysis, quantitative trait loci analysis, association mapping and marker assisted selection, not only for P. maxima, but also closely related species within the genus Pinctada.     

红原鸡全基因组中微卫星分布规律研究

本文对红原鸡Gallus gallus全基因组中微卫星数量及分布规律进行了分析,查找到l～6个碱基重复类型的微卫星序列共282728个,约占全基因组序列(1.1Gb)的0.49%,分布频率为1/3.89kb,微卫星序列的长度主要在12～70个碱基长度范围内。第1、2、3条染色体上微卫星分布频率较高,而32号染色体上无微卫星分布。不同类型微卫星中,单碱基重复类型数目最多,为184192个,占总数的65.1%;其次是四、二、三、五、六碱基重复单元序列,分别占到总数的12.8%、9.7%、7.2%、4.6%、0.8%。T、A、AT、GTTT、AAAC、G、C、ATTT、AC、GT、AAAT、ATT、AAC、AAT、GTT、AG、CT、CTTT、AAAG、GTTTT、AAACA、AAGG、CCTT是红原鸡基因组中最主要的微卫星重复类型。本研究为红原鸡微卫星标记的分离筛选、遗传多样性的研究以及不同物种微卫星的比较分析奠定了基础。     

Discovery and experimental analysis of microsatellites in an oil woody plant Camellia chekiangoleosa

Oil camellia trees are important woody plants for the production of high-quality cooking oil. On the contrary to their economic importance, their genetic and genomic resources are very limited, which greatly hamper the genetic studies on oil camellia trees. Microsatellites or simple sequence repeats (SSRs) have great value in many aspects of genetic analyses due to their high polymorphism and codominant inheritance. In this study, we report the large-scale development and characterization of SSR markers derived from genomic sequences of Camellia chekiangoleosa by high-throughput pyrosequencing technology. A total of 1,091,393 genomic shotgun reads were generated using Roche 454 FLX sequencer, the average read length was 319 bp, and the total sequence throughput was 347.9 Mb. These sequences were assembled into 35,315 contigs with total length of 14.8 Mb and the N50 contig size of 770 bp. By analyzing with microsatellite (MISA), a total of 5,844 perfect microsatellites were detected from the assembled sequences. Among them, tetranucleotide repeats were found to be the most frequent microsatellites in the genome of C. chekiangoleosa, and all the dominant repeat motifs for different types of SSRs were detected to be rich in A/T. Experimental analysis with 900 SSR primer pairs revealed that 66 % of them succeeded in PCR amplification. Further investigation with 345 SSR primer pairs showed that a relatively high percentage of primers amplified polymorphic loci (31.9 %). Experimental data also revealed that, overall, long microsatellite repeats (>20 bp) were more variable than the short ones (<20 bp) in the genome of oil camellia tree.     

Microsatellite marker identification using genome screening and restriction-ligation

We have identified a fast and easy method for finding microsatellite markers that utilizes genome screening with inter-simple sequence repeat (ISSR) primers to detect microsatellite regions and to obtain sequence information flanking one side of the microsatellites and a restriction-ligation technique with a specific adaptor to allow sequence walking to obtain sequence information flanking the other side of the microsatellites. Two main alternatives of the method (with or without cloning) are presented. We successfully utilized the method when identifying microsatellite markers for 21 bryophyte species, three algal species, and for the raccoon dog. The proportion of polymorphic markers equaled 95%. We observed that microsatellites are commonly found within the sequenced ISSR amplification products (54% in the present study), in which case specific primers can be identified for the microsatellite without a further restriction-ligation step. It is evident that the DNA regions amplified by ISSR markers commonly represent microsatellite hotspots. We propose that the identified method and the knowledge of the common presence of additional microsatellite repeats within ISSR amplification products are especially attractive to researchers who conduct small-scale microsatellite identification, such as researchers in population genetics and conservation biology.     

Genome‐wide microsatellite characterisation and marker development in Chenopodium quinoa

Microsatellites, as the tracts of repetitive DNA, are an essential constituent of the plant genome that holds important evolutionary significance, and have been extensively used to develop molecular makers for genetic analysis. To understand the microsatellite dynamics of quinoa genome and its relatives, in this study we performed a genome‐wide analysis of microsatellites in five Amaranthaceae species using available genome sequences. The results demonstrated that the microsatellites of the five Amaranthaceae species were characterised by relatively high proportions of mono‐, di‐ and trinucleotide repeats with A/T rich motifs, implying conservative organisation and composition of microsatellites in this family. Furthermore, a significant negative correlation between microsatellite frequencies and GC contents (r = ?.87) were observed. In total, 533,961 (89.57%) and 542,601 (89.86%) microsatellite loci could be used to develop simple sequence repeat (SSR) molecular markers, of which 7,178 were found to be polymorphic between the two sequenced quinoa cultivars, QQ74 and Real Blanca, through in silico PCR analysis. Finally, 15 SSR markers were randomly selected to validate their polymorphism across 12 quinoa accessions by wet‐lab PCR amplification. The newly developed genome‐wide SSR markers provide a useful resource for population genetics, gene mapping and molecular breeding studies in quinoa and beyond.