Mononucleotide Repeats Research Articles

Determining MSI status by a one-step PCR melting curve analysis of Chinese-specific mononucleotide repeats.

e15532 Background: Microsatellite instability (MSI) is a hypermutated phenotype primarily caused by the deficiency of DNA mismatch repair activity. Conventional MSI detection platforms are time consuming and need extra normal tissue. Due to the short DNA sequences of microsatellite loci in MSI samples, the method can be simplified to a one-step approach to differentiate MSI status by observing the melting curve. Herein a novel feasible MSI status detection method based on one step-PCR in single tumor tissue was developed, and validated in colorectal cancer samples. Methods: Specific microsatellite sites shorter than 12 bp in length (feasibility of fluorescence probes) were selected from public databases for analysis. In tumor tissues from 98 CRC patients with known MSI status, microsatellite sites with the highest receiver operating characteristic (ROC)-area under the ROC curve (AUC) were eventually selected for the final PCR panel design. The cut-off value of this assay was defined by the number of melting peaks in a specific Tm value range. An independent set of 199 tumor tissues from CRC patients with known MSI by Sanger sequencing was recruited to validate the performance of this novel PCR MSI kit. Results: The ROC-AUCs of the pre-selected eight mononucleotide repeats were 0.82, 0.93, 0.79, 0.78, 0.98, 0.95, 0.92, and 0.79. When 0 or 1 marker had a melting peak within the specified Tm value range, the sample was identified as "MSS/MSI-low (MSI-L)"; when 2 or more markers had melting peaks within the specified Tm value range, the sample was determined to be "MSI-high (MSI-H)". In the clinical sample set, compared with the results given by conventional MSI detection method, the sensitivity of the 8-gene panel for MSI-H was 99.4% (166/167), the specificity of the 8-gene panel was 100%, and the overall concordance reached to 99.5%. Conclusions: The novel PCR-MSI detection method using 8-gene panel developed in this study was feasible and accessible in clinical practice, and a large-scale clinical validation is in progress. [Table: see text]

A robust microsatellite instability detection model for unpaired colorectal cancer tissue samples.

Microsatellite instability (MSI) is a key biomarker for cancer immunotherapy and prognosis. Integration of MSI testing into a next-generation-sequencing (NGS) panel could save tissue sample, reduce turn-around time and cost, and provide MSI status and comprehensive genomic profiling in single test. We aimed to develop an MSI calling model to detect MSI status along with the NGS panel-based profiling test using tumor-only samples. From January 2019 to December 2020, a total of 174 colorectal cancer (CRC) patients were enrolled, including 31 MSI-high (MSI-H) and 143 microsatellite stability (MSS) cases. Among them, 56 paired tumor and normal samples (10 MSI-H and 46 MSS) were used for modeling, and another 118 tumor-only samples were used for validation. MSI polymerase chain reaction (MSI-PCR) was performed as the gold standard. A baseline was built for the selected microsatellite loci using the NGS data of 56 normal blood samples. An MSI detection model was constructed by analyzing the NGS data of tissue samples. The performance of the model was compared with the results of MSI-PCR. We first intersected the target genomic regions of the NGS panels used in this study to select common microsatellite loci. A total of 42 loci including 23 mononucleotide repeat sites and 19 longer repeat sites were candidates for modeling. As mononucleotide repeat sites are more sensitive and specific for detecting MSI status than sites with longer length motif and the mononucleotide repeat sites performed even better than the total sites, a model containing 23 mononucleotide repeat sites was constructed and named Colorectal Cancer Microsatellite Instability test (CRC-MSI). The model achieved 100% sensitivity and 100% specificity when compared with MSI-PCR in both training and validation sets. Furthermore, the CRC-MSI model was robust with the tumor content as low as 6%. In addition, 8 out of 10 MSI-H samples showed alternations in the four mismatch repair genes (MLH1, MSH2, MSH6, and PMS2). MSI status can be accurately determined along the targeted NGS panels using only tumor samples. The performance of mononucleotide repeat sites surpasses loci with longer repeat motif in MSI calling.

MLH1 Loss in Primary Prostate Cancer.

Among mismatch repair-deficient (MMRd) prostate cancers, loss of MLH1 is relatively uncommon and few cases have been reported in detail. Here, we describe the molecular features of two cases of primary prostate cancer with MLH1 loss detected by immunohistochemistry, and in one case, confirmed via transcriptomic profiling. Both cases were microsatellite stable on standard polymerase chain reaction (PCR)-based microsatellite instability (MSI) testing, but showed evidence of MSI on a newer PCR-based long mononucleotide repeat (LMR) assay and by next-generation sequencing. Germline testing was negative for Lynch syndrome-associated mutations in both cases. Targeted or whole-exome tumor sequencing using multiple commercial/academic platforms (Foundation, Tempus, JHU, and UW-OncoPlex) showed modestly elevated, though variable, tumor mutation burden estimates (2.3-10 mutations/Mb) consistent with MMRd, but without identifiable pathogenic single-nucleotide or indel mutations in MLH1. Copy-number analysis confirmed biallelic MLH1 loss in one case and monoallelic MLH1 loss in the second case, without evidence of MLH1 promoter hypermethylation in either. The second patient was treated with single-agent pembrolizumab and demonstrated a short-lived prostate-specific antigen response. These cases highlight the challenges in identifying MLH1-deficient prostate cancers using standard MSI testing and commercial sequencing panels, and support the utility of immunohistochemical assays and LMR- or sequencing-based MSI testing for detection of MMRd prostate cancers.

Genomic microsatellite characteristics analysis of Dysommaanguillare (Anguilliformes, Dysommidae), based on high-throughput sequencing technology.

Microsatellite loci were screened from the genomic data of Dysommaanguillare and their composition and distribution were analysed by bioinformatics for the first time. The results showed that 4,060,742 scaffolds with a total length of 1,562 Mb were obtained by high-throughput sequencing and 1,160,104 microsatellite loci were obtained by MISA screening, which were distributed on 770,294 scaffolds. The occurrence frequency and relative abundance were 28.57% and 743/Mb, respectively. Amongst the six complete microsatellite types, dinucleotide repeats accounted for the largest proportion (592,234, 51.05%), the highest occurrence frequency (14.58%) and the largest relative abundance (379.27/Mb). A total of 1488 microsatellite repeats were detected in the genome of D.anguillare, amongst which the hexanucleotide repeat motifs were the most abundant (608), followed by pentanucleotide repeat motifs (574), tetranucleotide repeat motifs (232), trinucleotide repeat motifs (59), dinucleotide repeat motifs (11) and mononucleotide repeat motifs (4). The abundance of microsatellites of the same repeat type decreased with the increase of copy numbers. Amongst the six types of nucleotide repeats, the preponderance of repeated motifs are A (191,390, 43.77%), CA (150,240, 25.37%), AAT (13,168, 14.05%), CACG (2,649, 8.14%), TAATG (119, 19.16%) and CCCTAA (190, 19.16%, 7.65%), respectively. The data of the number, distribution and abundance of different types of microsatellites in the genome of D.anguillare were obtained in this study, which would lay a foundation for the development of high-quality microsatellite markers of D.anguillare in the future.

Abstract 3954: Evaluation of long mononucleotide repeat markers for detection of microsatellite instability

Abstract Microsatellite instability (MSI) and mismatch repair immunohistochemistry are biomarkers of defective MMR (dMMR) that predict response to immune checkpoint inhibitor therapy in solid tumors. The microsatellites in the Promega MSI Analysis System Version 1.2 may not be ideal to detect MSI in certain cancer types. Long mononucleotide repeat (LMR) markers may improve detection of microsatellite instability in early colorectal lesions and non-colorectal cancers.In this study, we compared the performance of the current gold standard, MSI Analysis System Version 1.2 (Promega), which consists of 5 mononucleotide repeat markers (21-27 bases), with a new LMR kit (Promega), which consists of 4 markers from the MSI V1.2 panel and 4 LMR loci (52-60 bases). We studied five cohorts including 24 MMR proficient (pMMR) and 24 dMMR colorectal cancer (CRC) samples, 24 pMMR and 42 dMMR endometrial cancer (EC) samples, 12 dMMR prostate cancer (PC) samples, 22 MSI-high (MSI-H) samples of other cancer types, and 12 MSI-low (MSI-L) samples, where MMR status was confirmed by immunohistochemical (IHC) staining and/or MSI Analysis System Version 1.2.The specificity and sensitivity of the LMR MSI panel for dMMR detection were both 100% in CRC. The specificity of the MSI V1.2 and LMR MSI panels in EC was both 100%, and the sensitivity was 88% versus 98%, respectively. The 22 MSI-high (MSI-H) samples of other cancer types, these include cholangiocarcinoma, appendix, duodenal, pancreatic and gastric cancer, which were previously classified as MSI-H by using the MSI V1.2 panel were also classified as MSI-H by using the LMR MSI panel. Among 12 samples that were previously classified as MSI-L by the MSI V1.2 panel, 9 of the samples were classified as MSI-L, and 3 of the samples were diagnosed as MSI-H using the MSI LMR panel. The LMR panel has performed well on 80 samples over the past 6 months.The LMR MSI panel is highly concordant with the MSI V1.2 panel for dMMR detection in colorectal cancer and showed increased sensitivity in endometrial cancer. The LMR MSI panel showed improved dMMR detection in non-colorectal cancers. Citation Format: Suping Chen, John H. Lin, Aparna Pallavajjala, Tamara L. Lotan, Jeffery W. Bacher, James R. Eshleman. Evaluation of long mononucleotide repeat markers for detection of microsatellite instability. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3954.

Analysis of the chloroplast genome and phylogenetic evolution of Bidens pilosa

Chloroplast genomes for 3 Bidens plants endemic to China (Bidens bipinnata Linn., Bidens pilosa Linn., and Bidens alba var. radiata) have been sequenced, assembled and annotated in this study to distinguish their molecular characterization and phylogenetic relationships. The chloroplast genomes are in typical quadripartite structure with two inverted repeat regions separating a large single copy region and a small single copy region, and ranged from 151,599 to 154,478 bp in length. Similar number of SSRs and long repeats were found in Bidens, wherein mononucleotide repeats (A/T), forward and palindromic repeats were the most in abundance. Gene loss of clpP and psbD, IR expansion and contraction were detected in these Bidens plants. It seems that ndhE, ndhF, ndhG, and rpl32 from the Bidens plants were under positive selection while the majority of chloroplast genes were under purifying selection. Phylogenetic analysis revealed that 3 Bidens plants clustered together and further formed molophyletic clade with other Bidens species, indicating Bidens plants might be under radiation adaptive selection to the changing environment world-widely. Moreover, mutation hotspot analysis and in silico PCR analysis indicated that inter-genic regions of ndhD-ccsA, ndhI-ndhG, ndhF-rpl32, trnL_UAG-rpl32, ndhE-psaC, matK-rps16, rps2-atpI, cemA-petA, petN-psbM were candidate markers of molecular identification for Bidens plants. This study may provide useful information for genetic diversity analysis and molecular identification for Bidens species.

Bisulfite probing reveals DNA structural intricacies.

In recent decades, study of DNA structure has largely been focused on the interrelationships between nucleotides at the level of nearest neighbours. A little-utilized approach to probing structure on a larger scale is non-denaturing bisulfite modification of genomic DNA in conjunction with high-throughput sequencing. This technique revealed a marked gradient in reactivity increasing towards the 5' end of poly-dC:dG mononucleotide repeats as short as two base pairs, suggesting that access of the anion may be greater at these points due to positive-roll bending not predicted by existing models. Consistent with this, the 5' ends of these repeats are strikingly enriched at positions relative to the nucleosome dyad that bend towards the major groove, while their 3' ends tend to sit outside these areas. Mutation rates are also higher at the 5' ends of poly-dC:dG when CpG dinucleotides are excluded. These findings shed light on the mechanisms underlying bending/flexibility of the DNA double helix as well as the sequences that facilitate DNA packaging.

Comparison of microsatellite distribution in the genomes of Pteropus vampyrus and Miniopterus natalensis (Chiroptera)

BackgroundMicrosatellites are a ubiquitous occurrence in prokaryotic and eukaryotic genomes. Microsatellites have become one of the most popular classes of genetic markers due to their high reproducibility, multi-allelic nature, co-dominant mode of inheritance, abundance and wide genome coverage. We characterised microsatellites in the genomes and genes of two bat species, Pteropus vampyrus and Miniopterus natalensis. This characterisation was used for gene ontology analysis and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment of coding sequences (CDS).ResultsCompared to M. natalensis, the genome size of P. vampyrus is larger and contains more microsatellites, but the total diversity of both species is similar. Mononucleotide and dinucleotide repeats were the most diverse in the genome of the two species. In each bat species, the microsatellite bias was obvious. The microsatellites with the largest number of repeat motifs in P. vampyrus from mononucleotide to hexanucleotide were (A)n, (AC)n, (CAA)n, (AAAC)n, (AACAA)n and (AAACAA)n, with frequencies of 97.94%, 58.75%, 30.53%, 22.82%, 54.68% and 22.87%, respectively, while in M. natalensis were (A)n, (AC)n, (TAT)n, (TTTA)n, (AACAA)n and (GAGAGG)n, with of 92.00%, 34.08%, 40.36%, 21.83%, 25.42% and 12.79%, respectively. In both species, the diversity of microsatellites was highest in intergenic regions, followed by intronic, untranslated and exonic regions and lowest in coding regions. Location analysis indicated that microsatellites were mainly concentrated at both ends of the genes. Microsatellites in the CDS are thus subject to higher selective pressure. In the GO analysis, two unique GO terms were found only in P. vampyrus and M. natalensis, respectively. In KEGG enriched pathway, the biosynthesis of other secondary metabolites and metabolism of other amino acids in metabolism pathways were present only in M. natalensis. The combined biological process, cellular components and molecular function ontology are reflected in the GO analysis and six functional enrichments in KEGG annotation, suggesting advantageous mutations during species evolution.ConclusionsOur study gives a comparative characterization of the genomes of microsatellites composition in the two bat species. And also allow further study on the effect of microsatellites on gene function as well as provide an insight into the molecular basis for species adaptation to new and changing environments.

Genetic relationship of Pleione based on the chloroplast genome

Pleione (Orchidaceae) is a very famous horticultural plant with a high international reputation for its unique flower shape and abundant color. The small difference in morphological characteristics among Pleione species caused by weak reproductive isolation and easy hybridization makes the taxonomic status of individual species very confusing. Chloroplast (cp) genome information is of great significance for the study of plant phylogeny and taxonomy. In this study, the cp genomes of Pleione were sequenced and the complete cp structure and sequence characteristics of 19 species were compared and analyzed. The cp genome of Pleione species exhibited a conserved tetrad structure and each species encoded 135 protein-coding genes, 38 tRNA and 8 RNA genes. The cp genome sizes of 19 Pleione were 157964–159269 bp and the length of LSC, SSC and IR were 85953–87172 bp, 18499–18712 bp, 26459–26756 bp, respectively. Palindromic and forward repeats accounted for a high proportion and the SSRs were mainly mononucleotide repeats in Pleione. Analysis of chloroplast sequence differences indicated that the differences in coding regions were smaller than those in non-coding regions, and the variation in LSC and SSC regions was greater than that in IR regions. Phylogenetic analysis showed that all Pleione species inferred from the cp genome were clustered together and received high support. However, the genetic relationship of Pleione plants is different from the current update system of this genus. Therefore, the demarcation of Pleione interspecific relationships still needs further investigation due to the lack of sufficient evidence. The cp genome serves as valuable information for the identification of Pleione species and the study of phylogenetic relationships.

Genomically Stable Gastric Cancer Characterized by Hypomethylation in Wnt Signal Cascade

Introduction: Gastric cancer is divided into four subtypes by their molecular features linked with genetic alterations, e.g., Epstein-Barr virus (EBV), microsatellite instability-high (MSI-high), chromosomal instability (CIN), and genomically stable (GS), called as TCGA classification. In this study, we tried to clarify the epigenetic features of the four GC subtypes according to aberrant methylation status in 23 loci. Methods: A total of 98 gastric cancers and their normal gastric mucosa samples were included in this study. We divided gastric cancers into TCGA subtypes which were determined in line with MSI-high, EBV, CIN, to GS by their molecular features. The 13 loci of polymorphic microsatellite sequences were used to determine loss of heterogeneity for the detection of CIN. The MSI status was determined by three mononucleotide repeat markers. Infection of EBV was determined by recovering EBV BNRF1 sequence from genomic DNA collected from gastric cancers. Methylation status of 23 loci was investigated by the combined bisulfite restriction analysis. Status of other findings, e.g., KRAS mutations, HER2 expression status, and infection of helicobacter pylori were confirmed. Results: Gastric cancers were divided into MSI (13%), EBV (7%), CIN (53%), and GS (27%). By histological classification, poorly differentiated adenocarcinoma was more in tumors categorized in MSI-high, and GS and signet-ring cell carcinoma (sig) were more in GS. Among the 23 loci investigated their methylation status, 18 loci were significantly hypermethylated in caner tissues. An unsupervised clustering divided gastric cancers into two clusters and revealed that most GS tumors clustered together in a cluster that exhibited lower methylation levels, distinct from the other subtypes. The inter-variable clustering revealed that a cluster contained the three loci (SFRP2-region 1/2 and APC) belonging to the Wnt signal cascade (Wnt-associated loci). The mean methylation score of Wnt-associated loci was the lowest in GS tumors (MSI-high: 2.7 [95% confidence interval, 2.3–2.9]; EBV: 2.1 [1.2–3.1]; CIN: 2.4 [2.2–2.7]; GS: 1.3 [0.8–0.7]). In contrast, the mean methylation score of the other 15 loci was significantly higher in MSI-high, while that in GS was as same as that in EBV or CIN (MSI-high: 10.4 [8.3–12.4]; EBV: 5.7 [1.7–9.7]; CIN: 4.4 [3.6–5.1]; GS: 3.4 [2.2–4.6]). Additionally, the lower methylation score of Wnt-associated loci was observed only in sig tumors. Conclusions: GS subtype tumors have the potential to possess distinct signatures in DNA hypomethylation profiles in Wnt signaling pathway, especially in sig.

Analysis of SSR Information of Flower Transcriptome in Loquat

In order to provide scientific basis for studying the genetic diversity and molecular marker-assisted breeding of loquat, new molecular markers of loquat were developed by analyzing flower transcriptome data. In this study, MIcroSAtellite (MISA) and Blast2GO were used for SSR searching, screening, identification, and analysis of non-redundant Unigenes, and Primer 3 was used for SSR primer design. A search found that 28 617 Unigenes contained 44 622 SSR loci, and the frequency of occurrence of SSR loci was 47.51%, and the total average distribution distance was 4.87 kb. Mononucleotide and dinucleotide repeats were the main repeat types of SSR in the loquat flower transcriptome that the SSR of mononucleotide and dinucleotide repeats accounted for 52.09% and 32.83% of the total SSR, respectively. The dominant repeat motifs were A/T and AG/CT, which accounted for 50.94% and 26.70% of the mononucleotide repeats and dinucleotide respectively, and the 65.79% length of motif was concentrated in 12~20 bp. Gene Ontology (GO) functional classification showed that 28 617 loquat flower transcriptome Unigenes containing SSR loci were enriched in 51 GO terms of the 3 GO categories, and the biological process category involved the most GO terms (21). Primers for 34 301 SSR sequences were successfully designed, with a success rate of 76.87%. It is shown by the analysis that the flower transcriptome SSR loci had high frequency, high distribution density, and high polymorphism potential and could provide abundant repeat types. These could provide scientific basis for studying quantitative trait loci mapping, molecular marker-assisted breeding, genome evolution, genetic diversity analysis.

Constitutional Microsatellite Instability, Genotype, and Phenotype Correlations in Constitutional Mismatch Repair Deficiency

Constitutional mismatch repair deficiency (CMMRD) is a rare recessive childhood cancer predisposition syndrome caused by germline mismatch repair variants. Constitutional microsatellite instability (cMSI) is a CMMRD diagnostic hallmark and may associate with cancer risk. We quantified cMSI in a large CMMRD patient cohort to explore genotype-phenotype correlations using novel MSI markers selected for instability in blood. Three CMMRD, 1 Lynch syndrome, and 2 control blood samples were genome sequenced to >120× depth. A pilot cohort of 8 CMMRD and 38 control blood samples and a blinded cohort of 56 CMMRD, 8 suspected CMMRD, 40 Lynch syndrome, and 43 control blood samples were amplicon sequenced to 5000× depth. Sample cMSI score was calculated using a published method comparing microsatellite reference allele frequencies with 80 controls. Thirty-two mononucleotide repeats were selected from blood genome and pilot amplicon sequencing data. cMSI scoring using these MSI markers achieved 100% sensitivity (95% CI, 93.6%-100.0%) and specificity (95% CI 97.9%-100.0%), was reproducible, and was superior to an established tumor MSI marker panel. Lower cMSI scores were found in patients with CMMRD with MSH6 deficiency and patients with at least 1 mismatch repair missense variant, and patients with biallelic truncating/copy number variants had higher scores. cMSI score did not correlate with age at first tumor. We present an inexpensive and scalable cMSI assay that enhances CMMRD detection relative to existing methods. cMSI score is associated with mismatch repair genotype but not phenotype, suggesting it is not a useful predictor of cancer risk.

EST-SSR Markers' Development Based on RNA-Sequencing and Their Application in Population Genetic Structure and Diversity Analysis of Eleusine indica in China.

Goosegrass (Eleusine indica) is one of the worst agricultural weeds in China. Molecular markers were developed for genetic diversity and population structure analyses. In this study, we identified 8391 expressed sequence tag-simple sequence repeat (EST-SSR) markers from the de novo assembled unigenes of E. indica. Mononucleotides were the most abundant type of repeats (3591, 42.79%), followed by trinucleotides (3162, 37.68%). The most dominant mononucleotide and trinucleotide repeat motifs were A/T (3406, 40.59%) and AAT/ATT (103, 1.5%), respectively. Fourteen pairs of EST-SSR primers were verified and used to analyze the genetic diversity and population structure of 59 goosegrass populations. A total of 49 alleles were amplified, with the number of alleles (Na) ranging from two to eleven per locus, and the effective number of alleles (Ne) ranged from 1.07 to 4.53. The average polymorphic information content (PIC) was 0.36. Genetic structure analysis (K = 2) and principal coordinate analysis divided 59 E. indica populations into two groups in a manner similar to the unweighted pair-group method (Dice genetic similarity coefficient = 0.700). This study developed a set of EST-SSR markers in E. indica and successfully analyzed the diversity and population genetic structures of 59 E. indica populations in China.

Transcriptome analysis and development of EST-SSR markers in Anoectochilus emeiensis (Orchidaceae).

Anoectochilus emeiensis K. Y. Lang, together with other Anoectochilus species, has long been used as the main source of many traditional Chinese medicines. Owing to the shortcomings of molecular markers, the study of the genetic diversity and medicinal component synthesis mechanism of the endemic Anoectochilus species has been delayed. In this study, I carried out a transcriptome analysis of A. emeiensis. A total of 78,381 unigenes were assembled from 64.2 million reads, and 47,541 (60.65%) unigenes were matched to known proteins in the public databases. Then, 9284 expressed sequence tag-derived simple sequence repeats (EST-SSRs) were identified, and the frequency of SSRs in the A. emeiensis transcriptome was 9.88%. Trinucleotide repeats (3699, 39.84%) were the most common type, followed by dinucleotide (3251, 35.02%) and mononucleotide (1750, 18.85%) repeats. Based on the SSR sequence, 6683 primer pairs were successfully designed, 40 primer pairs were randomly selected, and 10 primer pairs were identified as polymorphic loci from 186 individuals of A. emeiensis. The EST-SSR markers examined in this study will be informative for future population genetic studies of A. emeiensis.

Comparative phylogenetic analysis of oolong tea (Phoenix Dancong tea) using complete chloroplast genome sequences

Phoenix Dancong tea, a variety of oolong tea, is produced in Chaozhou, Guangdong Province, China, and is characterized by numerous hybridizations and polyploidization. To assess the genetic diversity and phylogenetic relationships among Phoenix Dancong tea and other oolong teas, an integrated circular chloroplast genome was constructed for thirty species of Phoenix Dancong tea from Chaozhou. The genome of Phoenix dancong tea is a circular molecule of 157,041–157,137 bp, with a pair of inverted repeats (26,072-26,610 bp each) separated by a large single copy (86,615-86,658 bp) and small single copy (18,264-18,284 bp). A total of 135 unique genes were encoded, including 90 protein coding genes, 37 tRNAs and 8 rRNAs. A comparative analysis with the other seven species in the oolong tea family that have been sequenced to date revealed similarities in structural organization, gene content and arrangement. Repeated sequence analysis identified 17–23 tandem repeats, 20–24 forward repeats and 25–27 palindromic repeats. Additionally, a total of 65–70 simple sequence repeats were detected, with mononucleotide repeats being the most common. Phylogenetic analyses showed that Phoenix Dancong tea and Fujian oolong tea were clustered with other cultivated Camellia sinensis in the genus Camellia of the family Theaceae, while the two oolong tea species were relatively independently cross-embedded in the genus, Camellia. Close genetic relationships were observed between Phoenix Dancong tea and other oolong teas, and the overall chloroplast genomes of oolong tea showed patterns with low variations and conserved evolution. The availability of Phoenix Dancong tea chloroplast genomes not only elucidated the relationship among oolong teas from different origins in Guangdong and Fujian but also provided valuable genetic resources to assist further molecular studies on the taxonomic and phylogenomic resolution of the genus Camellia.

Microsatellite signature analysis of twenty-one virophage genomes of the family Lavidaviridae

Microsatellites or Simple Sequence Repeats (SSRs) are short motif repeat sequences constituting the most hypervariable regions of genomes. Present study extracts and analyzes the SSRs from genomes of 21 virophages. Genomic sequences were retrieved from NCBI and the microsatellite data was extracted through MISA web server. Phylogenetic analysis was performed by using MAFFT and MEGAX as per standardized protocols. The virophages have a circular/linear ds DNA genome of ~17-30 kb size. The GC% of genomes ranged from 26.8 (PSAV13) to 51.1 (PSAV12). A total of 3664 SSRs and 488 cSSR were observed with an average incidence of 174 and 23 respectively. The total SSR incidence in a genome ranged from 120 (PSAV19) to 264 (PSAV14). The cSSR (compound SSR) incidence ranged from 8 (PSAV12) to 47 (PSAV14). Mono-nucleotide repeats are the most incident microsatellites (1129 SSRs) followed by di-nucleotide (1036 SSRs) and tri-nucleotide repeats (368 SSRs). However, the same is not true for individual genomes. There are 14, 16 and 17 genomes which have no incidence of tetra-, penta- and hexa-nucleotide repeats respectively. Mono ‘A’ repeats having the maximum representation (average ~33 per genome) in mono-nucleotide repeats. For the di-nucleotide repeats, AT/TA motif had the highest frequency (average ~30) distantly followed by AG/GA; and CT/TC (average 5.6 & 5.5 respectively). A total of 1946 SSRs (76%) were found in the coding region. All genomes had a higher SSR density in non-coding as compared to the coding region. There are fifteen genomes which have at least one gene with no SSR. A total of 41 cSSRs with incidence across minimum of two virophages was observed. There were 12 cSSRs which had multiple presence within the same genome. The heat map of the genomes on one hand corroborates the phylogenetic tree with similar sequences (PSAV2, PSAV5, PSAV6, PSAV17 and PSAV18) being positioned together in the phylogenetic analysis while on the other hand it also highlights the diversity of the studied sequences. The conservation of cSSRs across multiple virophages highlights their potential as biomarkers.

Genome Survey Sequencing and Genetic Background Characterization of the Wolf Spider Pardosa pseudoannulata (Araneae: Lycosidae)

The wolf spider Pardosa pseudoannulata is a typical wandering spider and is frequently viewed as a biological indicator. However, published information on the P. pseudoannulata genome is limited. To explore the genomic structure of P. pseudoannulata, we performed a genome survey using high-throughput sequencing technologies at approximately 16.41X coverage depth. We obtained 67.48 Gb of sequence data, with an estimated genome size of 4.11 Gb and guanine plus cytosine (GC) content of 35.34%. The repeat ratio of the P. pseudoannulata genomic sequence was 79.16% and the heterozygote rate was 0.4%. By analysing the characteristics of the genome, 150,288 simple sequence repeats (SSRs) were identified. Among these SSRs, the mononucleotide repeat type was the most abundant (62.56% of total SSRs), followed by the dinucleotide (23.84%), trinucleotide (7.39%), tetranucleotide (3.94%), pentanucleotide (2.24%), and hexanucleotide (0.03%) repeat types. This is the first genome-wide characterization of P. pseudoannulata, which highlighted the genomic characteristics of low hybridization, high repetition, large size, and complex structure. The data obtained from our genome survey will not only aid the design of future P. pseudoannulata whole-genome sequencing projects but will also be valuable for further studies on genetic diversity and evolutionary analysis.

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

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

The Chloroplast Genome of the Lichen Photobiont Trebouxiophyceae sp. DW1 and Its Phylogenetic Implications.

Lichens are symbiotic associations of algae and fungi. The genetic mechanism of the symbiosis of lichens and the influence of symbiosis on the size and composition of the genomes of symbiotic algae have always been intriguing scientific questions explored by lichenologists. However, there were limited data on lichen genomes. Therefore, we isolated and purified a lichen symbiotic alga to obtain a single strain (Trebouxiophyceae sp. DW1), and then obtained its chloroplast genome information by next-generation sequencing (NGS). The chloroplast genome is 129,447 bp in length, and the GC content is 35.2%. Repetitive sequences with the length of 30–35 bp account for 1.27% of the total chloroplast genome. The simple sequence repeats are all mononucleotide repeats. Codon usage analysis showed that the genome tended to use codon ending in A/U. By comparing the length of different regions of Trebouxiophyceae genomes, we found that the changes in the length of exons, introns, and intergenic sequences affect the size of genomes. Trebouxiophyceae had an unstable chloroplast genome structure, with IRs repeatedly losing during evolution. Phylogenetic analysis showed that Trebouxiophyceae is paraphyletic, and Trebouxiophyceae sp. DW1 is sister to the clade of Koliella longiseta and Pabia signiensis.

A Mutation in the MYBL2-1 Gene Is Associated with Purple Pigmentation in Brassica oleracea.

Anthocyanins are well-known antioxidants that are beneficial for plants and consumers. Dihydroflavonol-4-reductase (DFR) is a key gene of anthocyanin biosynthesis, controlled by multiple transcription factors. Its expression can be enhanced by mutations in the negative regulator of anthocyanin biosynthesis myeloblastosis family transcription factor-like 2 (MYBL2). The expression profiles of the DFR gene were examined in 43 purple and green varieties of Brassica oleracea L., Brassica napus L., Brassica juncea L., and Brassica rapa L. MYBL2 gene expression was significantly reduced in purple varieties of B. oleracea, and green varieties of B. juncea. The MYBL2 gene sequences were screened for mutations that can affect pigmentation. Expression of the DFR gene was cultivar-specific, but in general it correlated with anthocyanin content and was higher in purple plants. Two single nucleotide polymorphysms (SNPs) were found at the beginning of the DNA-binding domain of MYBL2 gene in all purple varieties of B. oleracea. This mutation, leading to an amino acid substitution and the formation of a mononucleotide repeat (A)8, significantly affects RNA structure. No other noteworthy mutations were found in the MYBL2 gene in green varieties of B. oleracea and other studied species. These results bring new insights into the regulation of anthocyanin biosynthesis in genus Brassica and provide opportunities for generation of new purple varieties with precise mutations introduced via genetic engineering and CRISPR/Cas.