Tetranucleotide Motifs Research Articles

Abstract 3454: Cell-free DNA methylation and fragmentomic signatures identify tissue damage and predict cancer risk

Abstract Cell-free DNA (cfDNA) epigenetic and fragmentomic profiling has emerged as prominent non-invasive approaches for early cancer detection and subtyping. However, owing to difficulties in observing the early development of human malignancies, most cancer biomarker and evolution studies to date have primarily examined biologics following a diagnosis. Utilizing cfDNA as a screening tool for early disease detection requires assessment of blood plasma samples collected from asymptomatic individuals prior to the diagnosis of cancers. Here, we leverage the Ontario Health Study (OHS), a prospective longitudinal population cohort, to assess cfDNA profiles in blood plasma collected from participants that developed breast (n=476), prostate (n=342) and pancreatic (n=32) cancers throughout the study follow up time between 2009 and 2019. We performed cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) on baseline plasma samples from incident cancer cases up to eight years prior to diagnosis, in addition to matched controls (n=404) with no history of cancer through follow-up. Genome-wide plasma cfDNA differential methylation analysis revealed significantly hypermethylated regions, particularly among CpG island and shore regions, were predictive of early breast cancers, and overlapped hypermethylated regions in solid cancer tissues relative to adjacent normal tissues and peripheral blood leukocytes. Using a repeated cross-validation strategy we found that as few as 200 hypermethylated regions can identify individuals with breast cancer in blood up to seven years prior to diagnosis among discovery set samples (AUC=0.725) and are highly generalizable to samples processed in separate batches (AUC = 0.650). Remarkably, integrating biomarker hypermethylated regions with fragmentomic features including fragment length and 5’ tetranucleotide motif proportions further increased accuracy for detecting individuals with early prostate cancers (AUC=0.804). In addition, we observed shared genome-wide cfDNA methylome changes associated with aging and heavy alcohol consumption in cfDNA. We identified increased shedding of tissue-specific methylation markers from liver and pancreatic tissue among individuals consuming more than three drinks a week relative to non-drinkers, reflective of potential signatures of tissue damage. Citation Format: Nicholas Cheng, Kimberly Skead, Tom Oullette, Scott Bratman, Daniel De Carvalho, David Soave, Philip Awadalla. Cell-free DNA methylation and fragmentomic signatures identify tissue damage and predict cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3454.

Isolation and characterization of 17 polymorphic microsatellite loci with tri- and tetra-nucleotide repeat motifs for Ayu (Plecoglossus altivelis) using next-generation sequencing.

Ayu or sweetfish, Plecoglossus altivelis, an amphidromous fish ranging in the northwestern Pacific, is economically important inland fisheries and aquaculture resources. Genetic characterization of wild Ayu and derived culture seeds with competent molecular genetic markers is still insufficient for their sustainable use. Microsatellite DNA markers with larger repeat motifs (e.g. tri- and tetra-nucleotide motifs) are convenient and accurate compared with those having mono- and di-nucleotide motifs, but the latter motifs characterized most Ayu microsatellite markers developed previously. Here, we isolated and characterized 17 polymorphic microsatellite DNA markers with tri- and tetra-nucleotide repeat motif using next-generation sequencing. Alleles per locus varied from 6 to 23. The observed and expected heterozygosities ranged from 0.542 to1.000and 0.709to0.951, respectively. Polymorphic information content (PIC) of 15 out of the 17 loci were high (≧ 0.700), suggesting them to be highly informative. Twelve of the 17 loci were used for preliminary assignment test among three collections, and successfully allocated the examined fish to the original populations. The novel polymorphic microsatellite markers developed herein will be useful to examine the genetic diversity and population structure of wild Ayu and the effect of seed transplantation on native populations, providing a tool for conservation and sustainable adaptive management of this species.

DNA structural properties of DNA binding sites for 21 transcription factors in the mycobacterial genome.

Mycobacterium tuberculosis, the causative agent of tuberculosis, has evolved over time into a multidrug resistance strain that poses a serious global pandemic health threat. The ability to survive and remain dormant within the host macrophage relies on multiple transcription factors contributing to virulence. To date, very limited structural insights from crystallographic and NMR studies are available for TFs and TF-DNA binding events. Understanding the role of DNA structure in TF binding is critical to deciphering MTB pathogenicity and has yet to be resolved at the genome scale. In this work, we analyzed the compositional and conformational preference of 21 mycobacterial TFs, evident at their DNA binding sites, in local and global scales. Results suggest that most TFs prefer binding to genomic regions characterized by unique DNA structural signatures, namely, high electrostatic potential, narrow minor grooves, high propeller twist, helical twist, intrinsic curvature, and DNA rigidity compared to the flanking sequences. Additionally, preference for specific trinucleotide motifs, with clear periodic signals of tetranucleotide motifs, are observed in the vicinity of the TF-DNA interactions. Altogether, our study reports nuanced DNA shape and structural preferences of 21 TFs.

Systematic assessment of the flexibility of uracil damaged DNA

Uracil is a common DNA lesion which is recognized and removed by uracil DNA-glycosylase (UDG) as a part of the base excision repair pathway. Excision proceeds by base flipping, and UDG efficiency is thought to depend on the ease of deformability of the bases neighboring the lesion. We used molecular dynamics simulations to assess the flexibility of a large library of dsDNA strands, containing all tetranucleotide motifs with U:A, U:G, T:A or C:G base pairs. Our study demonstrates that uracil damaged DNA largely follows trends in flexibility of undamaged DNA. Measured bending persistence lengths, groove widths, step parameters and base flipping propensities demonstrate that uracil increases the flexibility of DNA, and that U:G base paired strands are more flexible than U:A strands. Certain sequence contexts are more deformable than others, with a key role for the 3′ base next to uracil. Flexibilities are large when this base is an A or G, and repressed for a C or T. A 5′ T adjacent to the uracil strongly promotes flexibility, but other 5′ bases are less influential. DNA bending is correlated to step deformations and base flipping, and bending aids flipping. Our study implies that the link between substrate flexibility and UDG efficiency is widely valid, helps explain why UDG prefers to bind U:G base paired strands, and suggests that the DNA bending angle of the UDG-substrate complex is optimal for base flipping. Communicated by Ramaswamy H. Sarma

Structural studies of SALL family protein zinc finger cluster domains in complex with DNA reveal preferential binding to an AATA tetranucleotide motif

The Spalt-like 4 transcription factor (SALL4) plays an essential role in controlling the pluripotent property of embryonic stem cells via binding to AT-rich regions of genomic DNA, but structural details on this binding interaction have not been fully characterized. Here, we present crystal structures of the zinc finger cluster 4 (ZFC4) domain of SALL4 (SALL4ZFC4) bound with different dsDNAs containing a conserved AT-rich motif. In the structures, two zinc fingers of SALL4ZFC4 recognize an AATA tetranucleotide. We also solved the DNA-bound structures of SALL3ZFC4 and SALL4ZFC1. These structures illuminate a common preference for the AATA tetranucleotide shared by ZFC4 of SALL1, SALL3, and SALL4. Furthermore, our cell biology experiments demonstrate that the DNA-binding activity is essential for SALL4 function as DNA-binding defective mutants of mouse Sall4 failed to repress aberrant gene expression in Sall4-/- mESCs. Thus, these analyses provide new insights into the mechanisms of action underlying SALL family proteins in controlling cell fate via preferential targeting to AT-rich sites within genomic DNA during cell differentiation.

The Landscape of Genome-Wide and Gender-Specific Microsatellites in Indo-Pacific Humpback Dolphin and Potential Applications in Cetacean Resource Investigation

Microsatellites are one of the important genome characterizations that can be a valuable resource for variety identification, genetic diversity, phylogenetic analysis, as well as comparative and conservation genomics research. Here, we developed comprehensive microsatellites through genome-wide mining for the threatened cetacean Indo-Pacific humpback dolphin (Sousa chinensis). We found 87,757 microsatellites with 2–6 bp nucleotide motifs, showing that about 32.5 microsatellites per megabase comprises microsatellites sequences. Approximately 97.8% of the markers developed in this study were consistent with the published identified markers. About 75.3% microsatellites were with dinucleotide motifs, followed by tetranucleotide motifs (17.4%), sharing the same composition pattern as other cetaceans. The microsatellites were not evenly distributed in the S. chinensis genome, mainly in non-coding regions, with only about 0.5% of the markers located in coding regions. The microsatellite-containing genes were mainly functionally enriched in the methylation process, probably demonstrating the potential impacts of microsatellites on biological functions. Polymorphic microsatellites were developed between different genders of S. chinensis, which was expected to lay the foundation for genetic diversity investigation in cetaceans. The specific markers for a male Indo-Pacific humpback dolphin will provide comprehensive and representative male candidate markers for sex identification, providing a potential biomolecular tool for further analysis of population structure and social behavior of wild populations, population trend evaluation, and species conservation management.

Characterization and microsatellite marker development for a common bark and ambrosia beetle associate, Geosmithia obscura.

Symbioses between Geosmithia fungi and wood‐boring and bark beetles seldom result in disease induction within the plant host. Yet, exceptions exist such as Geosmithia morbida, the causal agent of Thousand Cankers Disease (TCD) of walnuts and wingnuts, and Geosmithia sp. 41, the causal agent of Foamy Bark Canker disease of oaks. Isolates of G. obscura were recovered from black walnut trees in eastern Tennessee and at least one isolate induced cankers following artificial inoculation. Due to the putative pathogenicity and lack of recovery of G. obscura from natural lesions, a molecular diagnostic screening tool was developed using microsatellite markers mined from the G. obscura genome. A total of 3256 candidate microsatellite markers were identified (2236, 789, 137 di‐, tri‐, and tetranucleotide motifs, respectively), with 2011, 703, 101 di‐, tri‐, and tetranucleotide motifs, respectively, containing markers with primers. From these, 75 microsatellite markers were randomly selected, screened, and optimized, resulting in 28 polymorphic markers that yielded single, consistently recovered bands, which were used in downstream analyses. Five of these microsatellite markers were found to be specific to G. obscura and did not cross‐amplify into other, closely related species. Although the remaining tested markers could be useful, they cross‐amplified within different Geosmithia species, making them not reliable for G. obscura detection. Five novel microsatellite markers (GOBS9, GOBS10, GOBS41, GOBS43, and GOBS50) were developed based on the G. obscura genome. These species‐specific microsatellite markers are available as a tool for use in molecular diagnostics and can assist future surveillance studies.

Genome survey of Misgurnus anguillicaudatus to identify genomic information, simple sequence repeat (SSR) markers, and mitochondrial genome.

The dojo loach Misgurnus anguillicaudatus is an important economic species in Asia because of its nutritional value and broad environmental adaptability. Despite its economic importance, genomic data for M. anguillicaudatus is currently unavailable. In the present study, we conducted a genome survey of M. anguillicaudatus using next-generation sequencing technology. Its genome size was estimated to be 1105.97Mb by using K-mer analysis, and its heterozygosity ratio, repeat sequence content, GC content were 1.45%, 58.98%, and 38.03%, respectively. A total of 376,357 microsatellite motifs were identified, and mononucleotides, with a frequency of 42.57%, were the most frequently repeated motifs, followed by 40.83% dinucleotide, 7.49% trinucleotide, 8.09% tetranucleotide, and 0.91% pentanucleotide motifs. The AC/GT, AAT/ATT, and ACAG/CTGT repeats were the most abundant motifs among dinucleotide, trinucleotide, and tetranucleotide motifs, respectively. Besides,the complete mitochondrial genome was sequenced. Based on the Maximum Likelihood and Bayesian inference analyses, M. anguillicaudatus yingde in this study was the "introgressed" mitochondrial type. Seventy microsatellite loci were randomly selected from detected SSR loci to test polymorphic, of which, 20 microsatellite loci were assessed in 30 individuals from a wild population. The number of alleles (Na), observed heterozygosity (Ho), and expected heterozygosity (He) per locus ranged from 7 to 19, 0.400 to 0.933, and 0.752 to 0.938, respectively. All 20 loci were highly informative (PIC > 0.700). Eight loci deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.05). This is the first report of genome survey sequencing in M. anguillicaudatus, genome information, mitochondrial genome, andmicrosatellite markers will be valuable for further studies on population genetic analysis, natural resource conservation, and molecular marker-assisted selective breeding.

Genotyping pigs of Ukrainian breeds by microsatellite loci with a tetranucleotide motif

The aim of the research was genotyping animals of Ukrainian pig breeds according to microsatellite loci of the genome for further creation and implementation of the methodology for determining gene complexes of pig adaptability. Research methods: zootechnical – the analysis of genealogy and the productivity indexes of animals of different breeds; bioinformation - work with databases of the primary genome sequences for the design of oligonucleotide primers; molecular genetic - nucleic acid extraction, the determination of purity and concentration of DNA preparations, the amplification of microsatellite loci in PCR with subsequent electrophoretic separation of the amplification products in denaturing gel; statistical – the determination of population-genetic parameters on the basis of typing data on microsatellite loci, the relationship of microsatellite loci with performance indexes, the search for adaptive complexes. It was carried out genotyping animals of the Ukrainian meat breed in SE “RF Elita MIW named after V. M. Remeslo NAAS "of Kyiv region for 6 microsatellite loci with tetranucleotide repeats, a panel of primers of its own design. It was found the fact that the average number of alleles per locus was 5.2 alleles. The level of actual heterozygosity was 0.830, while the expected heterozygosity was 0.780. However, this difference is not significant. DNA typing animals for 6 loci of microsatellite DNA of 17 heads of Poltava meat breed of SE "EB Nadiia" was carried out. As a result of genotyping, the average number of alleles per locus was 3.8. The actual and expected heterozygosity levels were 0.680 and 0.730, respectively. By loci of microsatellite DNA, we found in animals of Myrhorod breed: for locus FH3628 – 5 alleles of 200-228 nucleotides, FH1865 – 5 alleles (size from 109 to 129 nucleotides), FH4219 – 2 alleles of 80 and 92 nucleotides, FH1885 – 4 alleles from 205 up to 213 nucleotides, FH3764 – 2 alleles 138 and 146 nucleotides, and for FH4231 – 2 alleles 116 and 120 nucleotides. The level of actual heterozygosity was 0.860, while the expected heterozygosity was 0.810. Thus, in three breeds of Ukrainian selection for 6 loci of microsatellite DNA with tetranucleotide repeat, the level of actual heterozygosity was in the range of 0.680 in animals of Poltava meat breed to 0.860 in animals of Myrhorod breed. The level of expected heterozygosity is 0.730 in animals of Poltava meat breed to 0.810 in Myrhorod breed. The high level of heterozygosity, as well as the average number of alleles per locus allows to search for alleles and genotypes associated with the adaptability of animals in breeds of pigs of Ukrainian selection. Key words: pigs, microsatellite loci, genome, average number of alleles, level of heterozygosity, population, variability.

Genome-wide mining of potentially-hypervariable microsatellites and validation of markers in Momordica charantia L.

Relatively large number of bitter melon microsatellite markers have been reported; however, only few resulted in successful PCR amplification and a small fraction shown polymorphisms. This limited chance of recovering polymorphic markers makes the primer screening a cost-demanding process. To test the hypothesis that microsatellites with longer motifs as well as shorter motifs repeated substantially shall have better prospects to be polymorphic, we performed a genome-wide microsatellite mining. We selected a sample of genome-wide microsatellites with prescribed motif lengths or satisfying a target repeat number, which were considered potentially-hyper variable, for primer designing and validation. Seventy five microsatellites satisfying these criteria were identified, of which 69 were validated through successful PCR amplification. Among them, 40 (53.33% of the markers identified) were polymorphic. This result showed a significantly higher success compared to our initial results of 51 (20.64%) polymorphic markers out of the 188 amplified when 247 previously reported markers were screened. The screening of two cultivars revealed that markers were efficient to identify up to three alleles. The characterization of these 69 new markers with 247 markers previously reported showed that di-nucleotide motifs were most abundant, followed by tri- and tetra-nucleotide motifs. TC motif markers were most polymorphic (12.08%) followed by AG and CT motifs (both 9.89%). Similarly, AGA (6.59%) and TATT (3.29%) were most polymorphic among the tri- and tetra-nucleotide motifs. These 69 hypervariable microsatellite markers along with 188 markers initially validated in this study shall be useful for phylogenetic analyses, studies of linkage, QTL, and association mapping in bitter melon.

Development and Cross-Amplification of Microsatellite Markers for Micrurus surinamensis (Elapidae)

Coral snakes have restricted and specialized diets, and their semifossorial and aquatic habits make them difficult to observe or collect. As a consequence, there is limited information about many aspects of their biology, including genetic diversity. Microsatellite markers have been used to study genetic variability in nonmodel species, such as snakes. The aim of this work was to develop primers for microsatellite regions of the genome of Micrurus surinamensis, to test transferability potential to Micrurus lemniscatus and Micrurus paraensis, and to analyze the genetic diversity of those species. DNA of one specimen of M. surinamensis was sequenced on the MiSeq platform, and sequences were searched for microsatellite regions with 10 primers designed for regions having tetranucleotide motifs. Micrurus surinamensis (n = 12 individuals) presented from 5 to 13 alleles and expected heterozygosity ranged from 0.741 to 0.939. Eight markers were successfully transferred to M. lemniscatus (n = 38) and five to M. paraensis (n = 13). The probability of exclusion of paternity ranged from 0.997 to 0.999 for species, and the probability of combined identity was lower in M. surinamensis (8.41 · 10-9). These are the first microsatellite markers to be used for studies of coral snake population genetics and conservation.

A gene regulatory network for antenna size control in carbon dioxide-deprived Chlamydomonas reinhardtii cells.

In green microalgae, prolonged exposure to inorganic carbon depletion requires long-term acclimation responses, involving modulated gene expression and the adjustment of photosynthetic activity to the prevailing supply of carbon dioxide. Here, we describe a microalgal regulatory cycle that adjusts the light-harvesting capacity at photosystem II (PSII) to the prevailing supply of carbon dioxide in Chlamydomonas (Chlamydomonas reinhardtii). It engages low carbon dioxide response factor (LCRF), a member of the squamosa promoter-binding protein (SBP) family of transcription factors, and the previously characterized cytosolic translation repressor nucleic acid-binding protein 1 (NAB1). LCRF combines a DNA-binding SBP domain with a conserved domain for protein-protein interaction. LCRF transcription is rapidly induced by carbon dioxide depletion. LCRF activates NAB1 transcription by specifically binding to tetranucleotide motifs present in its promoter. Accumulation of the NAB1 protein enhances translational repression of its prime target mRNA, encoding the PSII-associated major light-harvesting protein LHCBM6. The resulting truncation of the PSII antenna size helps maintaining a low excitation during carbon dioxide limitation. Analyses of low carbon dioxide acclimation in nuclear insertion mutants devoid of a functional LCRF gene confirm the essentiality of this novel transcription factor for the regulatory circuit.

Transcriptome Sequencing and Development of Novel Genic SSR Markers From Pistacia vera L.

In this study, we aimed to develop novel genic simple sequence repeat (eSSR) markers and to study phylogenetic relationship among Pistacia species. Transcriptome sequencing was performed in different tissues of Siirt and Atl cultivars of pistachio (Pistacia vera). A total of 37.5-Gb data were used in the assembly. The number of total contigs and unigenes was calculated as 98,831, and the length of N50 was 1,333 bp after assembly. A total of 14,308 dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide SSR motifs (4–17) were detected, and the most abundant SSR repeat types were trinucleotide (29.54%), dinucleotide (24.06%), hexanucleotide (20.67%), pentanucleotide (18.88%), and tetranucleotide (6.85%), respectively. Overall 250 primer pairs were designed randomly and tested in eight Pistacia species for amplification. Of them, 233 were generated polymerase chain reaction products in at least one of the Pistacia species. A total of 55 primer pairs that had amplifications in all tested Pistacia species were used to characterize 11 P. vera cultivars and 78 wild Pistacia genotypes belonging to nine Pistacia species (P. khinjuk, P. eurycarpa, P. atlantica, P. mutica, P. integerrima, P. chinensis, P. terebinthus, P. palaestina, and P. lentiscus). A total of 434 alleles were generated from 55 polymorphic eSSR loci with an average of 7.89 alleles per locus. The mean number of effective allele was 3.40 per locus. Polymorphism information content was 0.61, whereas observed (Ho) and expected heterozygosity (He) values were 0.39 and 0.65, respectively. UPGMA (unweighted pair-group method with arithmetic averages) and STRUCTURE analysis divided 89 Pistacia genotypes into seven populations. The closest species to P. vera was P. khinjuk. P. eurycarpa was closer P. atlantica than P. khinjuk. P. atlantica–P. mutica and P. terebinthus–P. palaestina pairs of species were not clearly separated from each other, and they were suggested as the same species. The present study demonstrated that eSSR markers can be used in the characterization and phylogenetic analysis of Pistacia species and cultivars, as well as genetic linkage mapping and QTL (quantitative trait locus) analysis.

Can Ferret Badgers (Melogale moschata) Cross the Rabies Epidemic Boundary in Western Taiwan?

Taiwan was considered rabies-free for > 50 years until the disease reemerged in its ferret badger population (Melogale moschata) in 2013. Although it is still expanding slowly northward in eastern Taiwan, its spread in the west has thus far been halted by the Da-An River. To prevent diffusion of rabies, an immunization zone should be established along the epidemic boundary before further rabies expansion crosses it. This study developed 19 microsatellite markers with tri- or tetra-nucleotide motifs specifically for ferret badgers using next generation sequencing, and we used these markers to evaluate level of gene flow across the Da-An River. Low levels of genetic differentiation and high levels of gene flow across the river indicated that the Da-An River was not a permanent barrier to rabies. Rabies may eventually be able to expand across this temporary boundary. We suggest that an immunization zone should be established along the Da-An River as soon as possible.

Whole genome survey analysis and microsatellite motif identification of Sebastiscus marmoratus.

The marbled rockfish Sebastiscus marmoratus is an ecologically and economically important marine fish species distributed along the northwestern Pacific coast from Japan to the Philippines. Here, next-generation sequencing was used to generate a whole genome survey dataset to provide fundamental information of its genome and develop genome-wide microsatellite markers for S. marmoratus. The genome size of S. marmoratus was estimated as approximate 800 Mb by using K-mer analyses, and its heterozygosity ratio and repeat sequence ratio were 0.17% and 39.65%, respectively. The preliminary assembled genome was nearly 609 Mb with GC content of 41.3%, and the data were used to develop microsatellite markers. A total of 191,592 microsatellite motifs were identified. The most frequent repeat motif was dinucleotide with a frequency of 76.10%, followed by 19.63% trinucleotide, 3.91% tetranucleotide, and 0.36% pentanucleotide motifs. The AC, GAG, and ATAG repeats were the most abundant motifs of dinucleotide, trinucleotide, and tetranucleotide motifs, respectively. In summary, a wide range of candidate microsatellite markers were identified and characterized in the present study using genome survey analysis. High-quality whole genome sequence based on the “Illumina+PacBio+Hi-C” strategy is warranted for further comparative genomics and evolutionary biology studies in this species.

A Multivariate Data Analytics Approach Revealed No Footprint of APOBEC3 Proteins in Hepatitis B Virus Genome

The human genome encodes for a family of editing enzymes known as APOBEC3 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like3). Several member of this family such as APOBEC3G, APOBEC3F and APOBEC3H haplotype II are active against viruses such as HIV. These enzymes induce C-to-U mutations in the negative strand of viral genomes, which leads to multiple G-to-A changes, referred to as ‘hypermutation’. Mutation by these enzymes are sequence context dependent in HIV genome, in which APOBEC3G preferentially mutates G within GG, TGG and TGGG content while other members mutate G within GA, TGA and TGAA. However, the same context between these enzymes and HBV virus have never been explored. In this study, we aim to identify mutational footprint of APOBEC3 enzymes in HBV genome. To do this, we perform a multivariable data analytics technique to investigate the motif preference and possible sequence hierarchy of mutation by APOBEC3 enzymes in a large number of full genome HBV sequences from a wide range of naturally infected patients. This enables us to differentiate between normal and hypermutated sequences in terms of the representation of mono- to tetra-nucleotide motifs and allows us to identify motifs associated with hypermutation by different APOBEC3 enzymes in the HBV genomes. Our analyses reveal that either APOPEC3 enzymes are not active against HBV virus or inducing G-to-A mutations by these enzymes are not sequence context dependent in HBV genome. Funding Statement: This work was supported by grants to HAR. HAR is currently supported by the UNSW Scientia Fellowship. Declaration of Interests: The authors declare that they have no financial or non-financial competing interests. Ethics Approval Statement: The authors stated: Not applicable.

Genetically Independent Tetranucleotide to Hexanucleotide Core Motif SSR Markers for Identifying Lentinula edodes Cultivars

For the purpose of protecting the rights of Lentinula edodes breeders, we developed a new simple sequence repeat (SSR) marker set consisting only of genetically independent tetranucleotide or longer core motifs. Using available genome sequences for five L. edodes strains, we designed primers for 13 SSR markers that amplified polymorphic sequences in 20 L. edodes cultivars. We evaluated the independence of every possible marker pair based on genotype data. Consequently, eight genetically independent markers were selected. The polymorphic information content values of the markers ranged from 0.269 to 0.764, with an average of 0.409. The markers could distinguish among 20 L. edodes cultivars and produced highly repeatable and reproducible results. The markers developed in this study will enable the precise identification of L. edodes cultivars, and may be useful for protecting breeders’ rights.

In Silico development of new SSRs primer for aquaporin linked to drought tolerance in plants

ABSTRACTPlants are exposed to various stress factors including biotic and abiotic stresses. Drought is a limiting factor that minimizes the development and growth of several plants in arid and semi-arid regions. Stress response is usually occur at different levels, Morphological, physiological and biochemical while at the molecular level a large number of genes are involved. This study aims at developing a new SSR primer for aquaporin related to drought stress in plants. A total of 177 complete coding sequences (CDS) available in the NCBI database are downloaded. After analyzing with BLAST, 163 sequences are selected. 1294 SSR derived from these sequences are characterized with MISA and indicating that all sequences contained SSRs. The most abundant SSR has been tetra-nucleotide repeat motif (36%) and among all the tetra-nucleotide repeats, the motif AAAG/CTTT was the most common type, whereas in tri-nucleotide, the motif CCG/CGG has been the predominate type. By using Primer3, 1120 primer pairs are generated and after analyzing, only 735 non redundant primer pairs that present the good characteristics are selected. Among them, some of the pairs of primers are randomly selected and validated on DNA of various species using PCR and agarose gel.

Genome-scale portrait and evolutionary significance of human-specific core promoter tri- and tetranucleotide short tandem repeats

BackgroundWhile there is an ongoing trend to identify single nucleotide substitutions (SNSs) that are linked to inter/intra-species differences and disease phenotypes, short tandem repeats (STRs)/microsatellites may be of equal (if not more) importance in the above processes. Genes that contain STRs in their promoters have higher expression divergence compared to genes with fixed or no STRs in the gene promoters. In line with the above, recent reports indicate a role of repetitive sequences in the rise of young transcription start sites (TSSs) in human evolution.ResultsFollowing a comparative genomics study of all human protein-coding genes annotated in the GeneCards database, here we provide a genome-scale portrait of human-specific short- and medium-size (≥ 3-repeats) tri- and tetranucleotide STRs and STR motifs in the critical core promoter region between − 120 and + 1 to the TSS and evidence of skewing of this compartment in reference to the STRs that are not human-specific (Levene’s test p < 0.001). Twenty-five percent and 26% enrichment of human-specific transcripts was detected in the tri and tetra human-specific compartments (mid-p < 0.00002 and mid-p < 0.002, respectively).ConclusionOur findings provide the first evidence of genome-scale skewing of STRs at a specific region of the human genome and a link between a number of these STRs and TSS selection/transcript specificity. The STRs and genes listed here may have a role in the evolution and development of characteristics and phenotypes that are unique to the human species.

PCR optimization and allele distribution of SNAC1 gene coding region in rice (Oryza sativa L.)

Drought is the most serious abiotic stress that limits crop production in rain-fed environments. In this study genetic diversity of SNAC1 gene investigated in a collection of 83 diverse rice accessions from different geographical origins. Amplification of SNAC1 gene exons was performed by combined hot start-touchdown PCR protocol. The average number of alleles was 9.5 alleles. A total of 6 rare alleles were identified from exons of SNAC1. The average gene diversity index, PIC (Polymorphism information content) and Shannon value were 0.8518, 0.8343 and 2.0469, respectively. Evolutionary study based on Ewens-Watterson test showed that exon 1 of SNAC1 gene was probability under genetic drift. To identify potential SSR markers in SNAC1 gene sequence, SSR distributions within rice SNAC1 gene sequence were mined. Totally 15 microsatellite loci were detected which tri-nucleotide motifs (8) was being most abundant, followed by di- (6) and tetra-nucleotide (1) motifs. Maximum loci were found in 3′ UTR (5) (Untranslated regions), followed by in 5′ UTR (4) and coding sequences (3 for each exon). The present study revealed genetic divergence of SNAC1 gene coding regions and also mined SSR distributions within SNAC1 gene sequence and introduced an optimized PCR method. This information can be used for the development of drought tolerant rice varieties.