Abundant Repeat Research Articles

Comparative Genome Analysis Reveals Divergent Genome Size Evolution in a Carnivorous Plant Genus.

The C-value paradox remains incompletely resolved after >40 yr and is exemplified by 2,350-fold variation in genome sizes of flowering plants. The carnivorous Lentibulariaceae genus Genlisea, displaying a 25-fold range of genome sizes, is a promising subject to study mechanisms and consequences of evolutionary genome size variation. Applying genomic, phylogenetic, and cytogenetic approaches, we uncovered bidirectional genome size evolution within the genus Genlisea. The Genlisea nigrocaulis Steyerm. genome (86 Mbp) has probably shrunk by retroelement silencing and deletion-biased double-strand break (DSB) repair, from an ancestral size of 400 to 800 Mbp to become one of the smallest among flowering plants. The G. hispidula Stapf genome has expanded by whole-genome duplication (WGD) and retrotransposition to 1550 Mbp. Genlisea hispidula became allotetraploid after the split from the G. nigrocaulis clade ∼29 Ma. Genlisea pygmaea A. St.-Hil. (179 Mbp), a close relative of G. nigrocaulis, proved to be a recent (auto)tetraploid. Our analyses suggest a common ancestor of the genus Genlisea with an intermediate 1C value (400-800 Mbp) and subsequent rapid genome size evolution in opposite directions. Many abundant repeats of the larger genome are absent in the smaller, casting doubt on their functionality for the organism, while recurrent WGD seems to safeguard against the loss of essential elements in the face of genome shrinkage. We cannot identify any consistent differences in habitat or life strategy that correlate with genome size changes, raising the possibility that these changes may be selectively neutral.

Comparative analysis of microsatellites and compound microsatellites in T4-like viruses

Microsatellites or simple sequence repeats (SSRs) are known to present ubiquitously in genomes of eukaryotes and prokaryotes, as well as viruses. A comprehensive analysis of microsatellites and compound microsatellites (CM) was performed for 67 T4-like bacteriophage genomes. We found that the number of repeats was generally proportional to the size of the genome. CM were more abundant in genic regions, while their relative abundance was higher in intergenic regions. Meanwhile, the number of CM rapidly decreased with the increase of complexity but gradually increased with higher dMAX (maximum distance between any two adjacent microsatellites). (A)n/(T)n, (AT)n/(TA)n and (AAG)n were the most abundant repeats of mono-, di- and trinucleotide microsatellites, respectively. The number of microsatellites in reference sequences was significantly lower than that in corresponding random sequences. This result was mainly attributed to mono- and dinucleotide repeats which hardly exceeded 6bp in T4-like viruses. These observations may be helpful to understand the distribution of microsatellites and viral genetic diversity in T4-like viruses.

Molecular marker development and genetic diversity exploration by RNA-seq in Platycodon grandiflorum.

Platycodon grandiflorum, generally known as the bellflower or balloon flower, is the only species in the genus Platycodon of the family Campanulaceae. Platycodon plants have been traditionally used as a medicinal crop in East Asia for their antiphlogistic, antitussive, and expectorant properties. Despite these practical uses, marker-assisted selection and molecular breeding in platycodons have lagged due to the lack of genetic information on this genus. In this study, we performed RNA-seq analysis of three platycodon accessions to develop molecular markers and explore genetic diversity. First, genic simple sequence repeats (SSRs) were retrieved and compared; dinucleotide motifs were the most abundant repeats (39%-40%) followed by trinucleotide (25%-31%), tetranucleotide (1.5%-1.9%), and pentanucleotide (0.3%-1.0%) repeats. The result of in silico SSR analysis, three SSR markers were detected and showed possibility to distinguish three platycodon accessions. After several filtering procedures, 180 single nucleotide polymorphisms (SNPs) were used to design 40 cleaved amplified polymorphic sequence (CAPS) markers. Twelve of these PCR-based markers were validated as highly polymorphic and utilized to investigate genetic diversity in 21 platycodon accessions collected from various regions of South Korea. Collectively, the 12 markers yielded 35 alleles, with an average of 3 alleles per locus. Polymorphism information content (PIC) values ranged from 0.087 to 0.693, averaging 0.373 per locus. Since platycodon genetics have not been actively studied, the sequence information and the DNA markers generated from our research have the potential to contribute to further genetic improvements, genomic studies, and gene discovery in this genus.

A Complete and Accurate Ab Initio Repeat Finding Algorithm.

It has become clear that repetitive sequences have played multiple roles in eukaryotic genome evolution including increasing genetic diversity through mutation, changes in gene expression and facilitating generation of novel genes. However, identification of repetitive elements can be difficult in the ab initio manner. Currently, some classical ab initio tools of finding repeats have already presented and compared. The completeness and accuracy of detecting repeats of them are little pool. To this end, we proposed a new ab initio repeat finding tool, named HashRepeatFinder, which is based on hash index and word counting. Furthermore, we assessed the performances of HashRepeatFinder with other two famous tools, such as RepeatScout and Repeatfinder, in human genome data hg19. The results indicated the following three conclusions: (1) The completeness of HashRepeatFinder is the best one among these three compared tools in almost all chromosomes, especially in chr9 (8 times of RepeatScout, 10 times of Repeatfinder); (2) in terms of detecting large repeats, HashRepeatFinder also performed best in all chromosomes, especially in chr3 (24 times of RepeatScout and 250 times of Repeatfinder) and chr19 (12 times of RepeatScout and 60 times of Repeatfinder); (3) in terms of accuracy, HashRepeatFinder can merge the abundant repeats with high accuracy.

Transcriptome sequencing and development of EST-SSR markers in Pinus dabeshanensis, an endangered conifer endemic to China

Pinus dabeshanensis Cheng et Law (P. dabeshanensis) is an endangered endemic occurring along the border between Anhui Province and Hubei Province (China). This species is known from only four natural populations. However, few genomic studies have been conducted on this species. In the present study, a cDNA library of P. dabeshanensis needles was sequenced using Illumina HiSeq™ 2000 paired-end sequencing technology. By mining 42,248 non-redundant unigenes, 1966 expressed sequence tag–simple sequence repeats (EST-SSRs) derived from 1835 unigenes were identified. The most abundant repeat motif was trinucleotide (41.6 %), followed by dinucleotide (23.25 %), mononucleotide (21.41 %), hexanucleotide (7.83 %), pentanucleotide (4.43 %) and tetranucleotide repeats (1.53 %). The top tri- and dinucleotide motifs included AGC/CTG and AT/AT, respectively. A total of 20,194 unigenes were assigned to three main GO categories with 153,373 GO terms. A KOG classification showed that 11,604 unigenes and 732 SSR-containing unigenes were assigned to 25 and 23 possible functional categories, respectively. Among the 1966 EST-SSRs, 431 primer pairs were successfully designed. After selecting 82 of these pairs at random for further validation, 44 pairs amplified unambiguous bands, 19 of which were polymorphic among 24 individuals of P. dabeshanensis. A total of 57 alleles were identified, varying from 2 to 5 alleles per locus with an average of 3.0. The observed and expected heterozygosity per locus ranged from 0 to 0.958 and from 0.082 to 0.759, respectively. The PIC values ranged from 0.077 to 0.707 with an average of 0.380. The generation of such large-scale sequence data in the present study provides a valuable resource for gene discovery and molecular marker development. Such markers will be robust tools for genomic research and breeding of P. dabeshanensis.

Mitochondrial SSRs and their utility in distinguishing wild species, CMS lines and maintainer lines in pigeonpea (Cajanus cajan L.)

AbstractAnalysis of the pigeonpea mitochondrial genome sequence identified 25 SSRs. Mononucleotide SSR motifs were the most abundant repeats followed by dinucleotide and trinucleotide repeats. Primer pairs could be designed for 24 SSRs, 23 of which were polymorphic amongst the 22 genotypes consisting of cytoplasmic male sterile (CMS or A) line, maintainer or B line and wild Cajanus species representing six different CMS systems viz., A1, A2, A4, A5, A6 and A8. These markers amplified a total of 107 alleles ranging from 2 to 10 with an average of 4.65 alleles per locus. The polymorphic information content for these markers ranged from 0.09 to 0.84 with an average of 0.52 per marker. Hence, the present study adds a novel set of 24 mitochondrial SSR markers to the markers repository in pigeonpea, which would be useful to distinguish the genotypes based on mitochondrial genome types in evolutionary and phylogenetic studies.

Comparative mitogenomic analysis of the superfamily Pentatomoidea (Insecta: Hemiptera: Heteroptera) and phylogenetic implications.

BackgroundInsect mitochondrial genomes (mitogenomes) are the most extensively used genetic marker for evolutionary and population genetics studies of insects. The Pentatomoidea superfamily is economically important and the largest superfamily within Pentatomomorpha with over 7,000 species. To better understand the diversity and evolution of pentatomoid species, we sequenced and annotated the mitogenomes of Eurydema gebleri and Rubiconia intermedia, and present the first comparative analysis of the 11 pentatomoid mitogenomes that have been sequenced to date.ResultsWe obtained the complete mitogenome of Eurydema gebleri (16,005 bp) and a nearly complete mitogenome of Rubiconia intermedia (14,967 bp). Our results show that gene content, gene arrangement, base composition, codon usage, and mitochondrial transcription termination factor sequences are highly conserved in pentatomoid species, especially for species in the same family. Evolutionary rate analyses of protein-coding genes reveal that the highest and lowest rates are found in atp8 and cox1 and distinctive evolutionary patterns are significantly correlated with the G + C content of genes. We inferred the secondary structures for two rRNA genes for eleven pentatomoid species, and identify some conserved motifs of RNA structures in Pentatomidea. All tRNA genes in pentatomoid mitogenomes have a canonical cloverleaf secondary structure, except for two tRNAs (trnS1 and trnV) which appear to lack the dihydrouridine arm. Regions that are A + T-rich have several distinct characteristics (e.g. size variation and abundant tandem repeats), and have potential as species or population level molecular markers. Phylogenetic analyses based on mitogenomic data strongly support the monophyly of Pentatomoidea, and the estimated phylogenetic relationships are: (Urostylididae + (Plataspidae + (Pentatomidae + (Cydnidae + (Dinidoridae + Tessaratomidae))))).ConclusionsThis comparative mitogenomic analysis sheds light on the architecture and evolution of mitogenomes in the superfamily Pentatomoidea. Mitogenomes can be effectively used to resolve phylogenetic relationships of pentatomomorphan insects at various taxonomic levels. Sequencing more mitogenomes at various taxonomic levels, particularly from closely related species, will improve the annotation accuracy of mitochondrial genes, as well as greatly enhance our understanding of mitogenomic evolution and phylogenetic relationships in pentatomoids.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1679-x) contains supplementary material, which is available to authorized users.

Genome wide survey and analysis of microsatellites in Tombusviridae family

Microsatellites are small repetitive sequences found in all kingdoms of life including viruses. Members of the Tombusviridae family severely affect growth of wide range of plants and reduce economic yield in diverse cropping systems worldwide. Here, we analyzed the nature and distribution of both simple and complex microsatellites present in complete genome of 47 species of Tombusviridae family. Our results showed, in all analyzed genomes, genome size and GC content had a weak influence on number, relative abundance and relative density of microsatellites, respectively. For each genome, dinucleotide repeats followed by mononucleotide repeats were found to be highly predominant. Mononucleotide repeats were found to be underrepresented in majority of Tombusviridae genomes. Poly G and Poly C repeats were more abundant as compared to poly A or poly T nucleotides. TG/GT and AG/CA was the most abundant dinucleotide repeat motif observed in Tombusviridae genomes. Repeats larger than trinucleotide were never found in these viral genomes. Comparative study of relative abundance and density of microsatellite among available RNA and DNA viral genomes indicated that simple repeats were lower than many RNA virus genomes. To our knowledge, this is the first analysis of microsatellites occurring in Tombusviridae genomes. Characterization of such variations in repeat sequences would be important in deciphering the origin, mutational processes, and role of repeat sequences in viral genomes.

Higher-order repeat structure in alpha satellite DNA occurs in New World monkeys and is not confined to hominoids.

Centromeres usually contain large amounts of tandem repeat DNA. Alpha satellite DNA (AS) is the most abundant tandem repeat DNA found in the centromeres of simian primates. The AS of humans contains sequences organized into higher-order repeat (HOR) structures, which are tandem arrays of larger repeat units consisting of multiple basic repeat units. HOR-carrying AS also occurs in other hominoids, but results reported to date for phylogenetically more remote taxa have been negative. Here we show direct evidence for clear HOR structures in AS of the owl monkey and common marmoset. These monkeys are New World monkey species that are located phylogenetically outside of hominoids. It is currently postulated that the presence of HOR structures in AS is unique to hominoids. Our results suggest that this view must be modified. A plausible explanation is that generation of HOR structures is a general event that occurs occasionally or frequently in primate centromeres, and that, in humans, HOR-carrying AS became predominant in the central region of the centromere. It is often difficult to assemble sequence reads of tandem repeat DNAs into accurate contig sequences; our careful sequencing strategy allowed us to overcome this problem.

Characterization of repetitive DNA landscape in wheat homeologous group 4 chromosomes.

BackgroundThe number and complexity of repetitive elements varies between species, being in general most represented in those with larger genomes. Combining the flow-sorted chromosome arms approach to genome analysis with second generation DNA sequencing technologies provides a unique opportunity to study the repetitive portion of each chromosome, enabling comparisons among them. Additionally, different sequencing approaches may produce different depth of insight to repeatome content and structure. In this work we analyze and characterize the repetitive sequences of Triticum aestivum cv. Chinese Spring homeologous group 4 chromosome arms, obtained through Roche 454 and Illumina sequencing technologies, hereinafter marked by subscripts 454 and I, respectively.Repetitive sequences were identified with the RepeatMasker software using the interspersed repeat database mips-REdat_v9.0p. The input sequences consisted of our 4DS454 and 4DL454 scaffolds and 4ASI, 4ALI, 4BSI, 4BLI, 4DSI and 4DLI contigs, downloaded from the International Wheat Genome Sequencing Consortium (IWGSC).ResultsRepetitive sequences content varied from 55% to 63% for all chromosome arm assemblies except for 4DLI, in which the repeat content was 38%. Transposable elements, small RNA, satellites, simple repeats and low complexity sequences were analyzed. SSR frequency was found one per 24 to 27 kb for all chromosome assemblies except 4DLI, where it was three times higher. Dinucleotides and trinucleotides were the most abundant SSR repeat units. (GA)n/(TC)n was the most abundant SSR except for 4DLI where the most frequently identified SSR was (CCG/CGG)n. Retrotransposons followed by DNA transposons were the most highly represented sequence repeats, mainly composed of CACTA/En-Spm and Gypsy superfamilies, respectively. This whole chromosome sequence analysis allowed identification of three new LTR retrotransposon families belonging to the Copia superfamily, one belonging to the Gypsy superfamily and two TRIM retrotransposon families. Their physical distribution in wheat genome was analyzed by fluorescent in situ hybridization (FISH) and one of them, the Carmen retrotransposon, was found specific for centromeric regions of all wheat chromosomes.ConclusionThe presented work is the first deep report of wheat repetitive sequences analyzed at the chromosome arm level, revealing the first insight into the repeatome of T. aestivum chromosomes of homeologous group 4.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1579-0) contains supplementary material, which is available to authorized users.

Gene discovery in the developing xylem tissue of a tropical timber tree species: Neolamarckia cadamba (Roxb.) Bosser (kelampayan)

A complementary DNA (cDNA) library was con- structed from the developing xylem tissues of Neolamarckia cadamba. A total of 10,368 single-pass sequences was gener- ated through high-throughput 5'-expressed sequence tag (EST) sequencing of the cDNA clones, and 6622 high- quality ESTs were obtained after removing the low-quality sequences; this gave approximately 3.17 Mb of data. Clustering of the high-quality ESTs revealed 4728 unigenes, consisting of 2100 consensus and 2628 singletons. A total of 2405 ESTs were successfully annotated with 7753 gene on- tology (GO) terms that distributed among three main GO cat- egories, which were biological processes (2333), molecular function (3056) and cellular component (2364). Simple se- quence repeat (SSR) mining revealed that the frequency of SSR in the N. cadamba EST database (NcbdEST) was 3.3 %, with the GCT/AGC motif being the most abundant repeat motif. The most abundant transcript with known func- tion found in this database was 60S ribosomal protein follow- ed by 40S ribosomal protein. Some of the important genes involved in xylogenesis and lignin biosynthesis were found in NcdbEST; these include tubulin genes, cellulose synthase (CesA), xyloglucan endotransglycosylase (XET), arabinogalactan, cinnamate 4-hydroxylase (C4H), caffeoyl- coenzyme A O-methyltransferase (CCoAOMT) and peroxi- dase. The data obtained from this study will provide a power- ful means for identifying mechanisms controlling wood for- mationpathwaysofkelampayanandsupplymanynew cloned genes for future endeavours to modify wood and fibre properties.

Development and Characterization of EST-SSR Markers in Taxodium ‘zhongshansa’

Taxodium ‘zhongshansa’, an extremely flood-tolerant tree species, has been widely planted in eastern China due to its environmental attributes. Although much progress has been made in understanding its physiological response to flooding stress, as well as its rapid propagation and variety selection, little is known regarding its molecular genetics due primarily to a lack of reliable molecular markers. In this paper, 108,692 expressed sequence tags (ESTs) with a total size of 69.3 Mb derived from T. ‘zhongshansa’ were analyzed. And 10,038 simple sequence repeat (SSR) loci were identified from 8137 SSR-containing EST sequences. The average SSR frequency in the transcriptome was one in 6.90 kb of EST sequences. The most abundant repeat type was mononucleotide (6581, 65.56 %), followed by trinucleotide (2246, 22.37 %) and dinucleotide (1080, 10.76 %). After filtering unqualified SSR loci, loci with mononucleotide repeats and compound SSRs, 1958 EST-SSR loci were selected to design SSR makers. To validate this set of SSR markers, 503 primer pairs were randomly selected to amplify across 12 genomic DNA templates from three Taxodium species. Two hundred fifty-seven (51.09 %) out of 503 primer pairs amplified the expected products, of which 81 and 176 could amplify polymorphic and monomorphic bands, respectively. The functional categorization of EST sequences containing randomly selected markers revealed that 264 (52.49 %) had homology with known proteins. This set of EST-SSR markers will provide a valuable genetic and genomic tool for further genetic research in Taxodium, such as genetic map construction, quantitative trait loci mapping, and marker assisted selection.

Rapid generation and analysis of expressed sequence tags to uncovering inflorescence secondary metabolism of Bougainvillea spectabilis ‘Speciosas’ by pyrosequencing

Bougainvilleas, are one of the unique plant families with an unusual class of pigments, known as betalains replaced the more common anthocyanins. However, little is known on the molecular mechanism on the mutual exclusion of anthocyanins and betalains. In order to explore the unique phenomena in Bougainvilleas, we used open inflorescences of B. spectabilis ‘Speciosas’ for transcriptome analysis by pyrosequencing, which is the first attempt to obtain the transcriptome of this specific plant. In this study, we obtained 111.9 M raw data, after assembling, 17,728 unigenes sequence with average length of 607 bp long were available, the annotation including Gene Ontology, COG/KOG and associated Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway maps were conducted. Important genes involved in flavonoid/Betalains/anthocyanins motablisms were identified including FLS/F3H, UGT, DFR, DODA, anthocyanidin reductase and Anthocyanidin 3-O-glucosyltransferase, as well as some transcriptional factors (Myb, YABBY, MADS-box, F-box, WD-40 and bHLH) in this study. The obtained unigenes which will provide a database for discovering genes involved in secondary metabolic synthesis pathway. The discovered genes can be used for future bioengineering of various secondary metabolites for health improvement, pigments, medicine and agricultural production, etc. Specially, these genes such as DFR, anthocyanidin reductase and anthocyanidin 3-O-glucosyltransferase, were detected in betalain-synthesizing plants, which is exciting for researchers. Furthermore, a total of 877 simple sequence repeat motifs (SSRs) were identified, among the perfect SSRs, the most abundant repeat units were mononucleotide repeats (61 %) with (A/T)n and the second one is tri-nucleotide repeats (24 %). The obtained EST-SSRs can be used for marker development which will be beneficial for the studies in the aspects of genetic diversity, evolution and phenotype variation of B. spectabilis ‘Speciosas’ as well as its related species.

Mining of single nucleotide polymorphism (SNP) and simple sequence repeats (SSRs) from EST tropical fruits

The advancement in genomics technology has produced vast amount of expressed sequencetags (ESTs) sequence from tropical fruits. These resources have increased the publicavailability of ESTs sequence from year after year. Therefore, this effort permits mining ofsingle nucleotide polymorphism (SNP) and simple sequence repeat (SSR) from EST tropicalfruits. SNP and SSR are types of molecular marker which commonly used in modern geneticanalysis for wide application such as diversity analysis, linkage analysis and association study.In this study, a small scale EST sequences from tropical fruits (pineapple, mango, coconut andbanana) were retrieved from dbEST database (www.ncbi.nlm.nih.gov/dbEST/ ) as of March2013. Various bioinformatics tools were applied for rapid discovery of SNP and SSR markerfrom EST sequences. We analyzed 31,920 unigenes (contigs and singletons) representing atotal of 77,418 ESTs from four tropical fruits for their potential use in developing SNP and SSRmarkers. A total of 13,709 EST-SNP were discovered while a total of 4853 EST-SSR werediscovered from these four tropical fruits. The most abundant EST-SSR repeat is fromtrinucleotide (15,957 repeats) followed by dinucleotide (13,797 repeats) and tetranucleotide(973 repeats). Here, 1738 primers from SNP while 2033 primers from SSR were passedthrough the setting criteria and were selected for validation using genotyping platform. Thisstudy not only serves as a resource for marker development in tropical fruits but can provide abetter insight into the selection of candidate genes of interest.

Chromosomal organizations of major repeat families on potato (Solanum tuberosum) and further exploring in its sequenced genome.

One of the most powerful technologies in unraveling the organization of a eukaryotic plant genome is high-resolution Fluorescent in situ hybridization of repeats and single copy DNA sequences on pachytene chromosomes. This technology allows the integration of physical mapping information with chromosomal positions, including centromeres, telomeres, nucleolar-organizing region, and euchromatin and heterochromatin. In this report, we established chromosomal positions of different repeat fractions of the potato genomic DNA (Cot100, Cot500 and Cot1000) on the chromosomes. We also analysed various repeat elements that are unique to potato including the moderately repetitive P5 and REP2 elements, where the REP2 is part of a larger Gypsy-type LTR retrotransposon and cover most chromosome regions, with some brighter fluorescing spots in the heterochromatin. The most abundant tandem repeat is the potato genomic repeat 1 that covers subtelomeric regions of most chromosome arms. Extensive multiple alignments of these repetitive sequences in the assembled RH89-039-16 potato BACs and the draft assembly of the DM1-3 516 R44 genome shed light on the conservation of these repeats within the potato genome. The consensus sequences thus obtained revealed the native complete transposable elements from which they were derived.

Accumulation of interspersed and sex-specific repeats in the non-recombining region of papaya sex chromosomes.

BackgroundThe papaya Y chromosome has undergone a degenerative expansion from its ancestral autosome, as a consequence of recombination suppression in the sex determining region of the sex chromosomes. The non-recombining feature led to the accumulation of repetitive sequences in the male- or hermaphrodite-specific regions of the Y or the Yh chromosome (MSY or HSY). Therefore, repeat composition and distribution in the sex determining region of papaya sex chromosomes would be informative to understand how these repetitive sequences might be involved in the early stages of sex chromosome evolution.ResultsDetailed composition of interspersed, sex-specific, and tandem repeats was analyzed from 8.1 megabases (Mb) HSY and 5.3 Mb corresponding X chromosomal regions. Approximately 77% of the HSY and 64% of the corresponding X region were occupied by repetitive sequences. Ty3-gypsy retrotransposons were the most abundant interspersed repeats in both regions. Comparative analysis of repetitive sequences between the sex determining region of papaya X chromosome and orthologous autosomal sequences of Vasconcellea monoica, a close relative of papaya lacking sex chromosomes, revealed distinctive differences in the accumulation of Ty3-Gypsy, suggesting that the evolution of the papaya sex determining region may accompany Ty3-Gypsy element accumulation. In total, 21 sex-specific repeats were identified from the sex determining region; 20 from the HSY and one from the X. Interestingly, most HSY-specific repeats were detected in two regions where the HSY expansion occurred, suggesting that the HSY expansion may result in the accumulation of sex-specific repeats or that HSY-specific repeats might play an important role in the HSY expansion. The analysis of simple sequence repeats (SSRs) revealed that longer SSRs were less abundant in the papaya sex determining region than the other chromosomal regions.ConclusionMajor repetitive elements were Ty3-gypsy retrotransposons in both the HSY and the corresponding X. Accumulation of Ty3-Gypsy retrotransposons in the sex determining region of papaya X chromosome was significantly higher than that in the corresponding region of V. monoica, suggesting that Ty3-Gypsy could be crucial for the expansion and evolution of the sex determining region in papaya. Most sex-specific repeats were located in the two HSY expansion regions.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-335) contains supplementary material, which is available to authorized users.

Investigation of optimum concentrations of betaine for improving the resolution of sequencing G-C rich DNA with trinucleotide repeats

To develop an optimal sequencing system which can improve the resolution of sequencing G-C rich DNA with abundant trinucleotide repeats by applying concentration gradients of betaine to the Sanger sequencing system. Concentration gradients of betaine were introduced into the sequencing system by taking the 5' terminal of Nogo-B cDNA (Am-Nogo-B) (G-C%=72%, without trinucleotide repeats) and 5' terminal of Huntingtin cDNA (Am-HTT) (G-C%=74%, with abundant CAG and CCG repeats) the results of sequencing were compared. The optimum concentration of betaine for sequencing Am-Nogo-B has differed from that for Am-HTT. Result of sequencing Am-Nogo-B has achieved the best quality when the concentration of betaine was at 0.8-1.2 mol/L, whereas the result of sequencing Am-HTT obtained the best quality when the concentration of betaine was at 1.6 -2.4 mol/L. The results were reproducible. G-C rich DNA with similar G-C% required different concentrations of betaine in the sequencing system due to base pair compositions. The sequencing system developed for improving the resolution of sequencing of G-C rich DNA with abundant trinucleotide repeats can be used as a reference for similar studies.

Development of genomic simple sequence repeat markers in opium poppy by next-generation sequencing

Opium poppy (Papaver somniferum L.) is an important pharmaceutical crop with very few genetic marker resources. To expand these resources, we sequenced genomic DNA using pyrosequencing technology and examined the DNA sequences for simple sequence repeats (SSRs). A total of 1,244,412 sequence reads were obtained covering 474 Mb. Approximately half of the reads (52 %) were assembled into 166,724 contigs representing 105 Mb of the opium poppy genome. A total of 23,283 non-redundant SSRs were identified in 18,944 contigs (11.3 % of total contigs). Trinucleotide and tetranucleotide repeats were the most abundant SSR repeats, accounting for 49.0 and 27.9 % of all SSRs, respectively. The AAG/TTC repeat was the most abundant trinucleotide repeat, representing 19.7 % of trinucleotide repeats. Other SSR repeat types were AT-rich. A total of 23,126 primer pairs (98.7 % of total SSRs) were designed to amplify SSRs. Fifty-three genomic SSR markers were tested in 37 opium poppy accessions and seven Papaver species for determination of polymorphism and transferability. Intraspecific polymorphism information content (PIC) values of the genomic SSR markers were intermediate, with an average 0.17, while the interspecific average PIC value was slightly higher, 0.19. All markers showed at least 88 % transferability among related species. This study increases sequence coverage of the opium poppy genome by sevenfold and the number of opium poppy-specific SSR markers by sixfold. This is the first report of the development of genomic SSR markers in opium poppy, and the genomic SSR markers developed in this study will be useful in diversity, identification, mapping and breeding studies in opium poppy.

Dynamic Alu methylation during normal development, aging, and tumorigenesis.

DNA methylation primarily occurs on CpG dinucleotides and plays an important role in transcriptional regulations during tissue development and cell differentiation. Over 25% of CpG dinucleotides in the human genome reside within Alu elements, the most abundant human repeats. The methylation of Alu elements is an important mechanism to suppress Alu transcription and subsequent retrotransposition. Decades of studies revealed that Alu methylation is highly dynamic during early development and aging. Recently, many environmental factors were shown to have a great impact on Alu methylation. In addition, aberrant Alu methylation has been documented to be an early event in many tumors and Alu methylation levels have been associated with tumor aggressiveness. The assessment of the Alu methylation has become an important approach for early diagnosis and/or prognosis of cancer. This review focuses on the dynamic Alu methylation during development, aging, and tumor genesis. The cause and consequence of Alu methylation changes will be discussed.

Medium-sized tandem repeats represent an abundant component of the Drosophila virilis genome

BackgroundPreviously, we developed a simple method for carrying out a restriction enzyme analysis of eukaryotic DNA in silico, based on the known DNA sequences of the genomes. This method allows the user to calculate lengths of all DNA fragments that are formed after a whole genome is digested at the theoretical recognition sites of a given restriction enzyme. A comparison of the observed peaks in distribution diagrams with the results from DNA cleavage using several restriction enzymes performed in vitro have shown good correspondence between the theoretical and experimental data in several cases. Here, we applied this approach to the annotated genome of Drosophila virilis which is extremely rich in various repeats.ResultsHere we explored the combined approach to perform the restriction analysis of D. virilis DNA. This approach enabled to reveal three abundant medium-sized tandem repeats within the D. virilis genome. While the 225 bp repeats were revealed previously in intergenic non-transcribed spacers between ribosomal genes of D. virilis, two other families comprised of 154 bp and 172 bp repeats were not described. Tandem Repeats Finder search demonstrated that 154 bp and 172 bp units are organized in multiple clusters in the genome of D. virilis. Characteristically, only 154 bp repeats derived from Helitron transposon are transcribed.ConclusionUsing in silico digestion in combination with conventional restriction analysis and sequencing of repeated DNA fragments enabled us to isolate and characterize three highly abundant families of medium-sized repeats present in the D. virilis genome. These repeats comprise a significant portion of the genome and may have important roles in genome function and structural integrity. Therefore, we demonstrated an approach which makes possible to investigate in detail the gross arrangement and expression of medium-sized repeats basing on sequencing data even in the case of incompletely assembled and/or annotated genomes.