Genome-wide SSR Markers in Bottle Gourd: Development, Characterization, Utilization in Assessment of Genetic Diversity of National Genebank of India and Synteny With Other Related Cucurbits

Lagenaria siceraria (Molina) Standley is an important cultivated crop with its immense importance in pharmaceutical industry and as vegetable. Its seed, root, stem, leaves, flower and fruit are used as an ointment for ailment of various diseases throughout Asia. Despite its worldwide importance,informative co-dominant microsatellite markers in the bottle gourd crop are very restricted, impeding geneticimprovement, cultivar identification and phylogenetic studies. Next generation sequencing has revolutionizedthe approaches for discovery, assessment and validation of molecular markers. We conducted a genome wideanalysis, for developing SSR markers by utilizing restriction site-associated DNA sequencing (RAD-Seq) data.By performing in silico mining of microsatellite repeat-motifs, we developed 45,066 perfect SSR markers. Ofwhich 203 markers were successfully validated and 101 (49.75%) polymorphic primer pairs were utilized for anin depth genetic diversity and population structure analysis of 96 accessions from the National Genebank ofIndia. Tetranucleotide repeats (∼34.3%) were the most prevalent followed by trinucleotide repeats (∼30.73%),further 21.03%, 9.6% and 4.3% of di-, penta- and hexa- nucleotide repeats in the bottle gourd genome. Syntenyof SSR markers on 11 bottle gourd linkage groups was correlated with the 7 chromosomes of cucumber(93.2%), 12 chromosomes of melon (87.4%) and 11 watermelon (90.8%). The generated SSR markers providea valuable tool for germplasm characterization, genetic linkage map construction, studying synteny, genediscovery and for breeding in bottle gourd and other cucurbits species.

Effects of Adenine Methylation on the Structure and Thermodynamic Stability of a DNA Minidumbbell

DNA methylation is a prevalent regulatory modification in prokaryotes and eukaryotes. N1-methyladenine (m1A) and N6-methyladenine (m6A) have been found to be capable of altering DNA structures via disturbing Watson–Crick base pairing. However, little has been known about their influences on non-B DNA structures, which are associated with genetic instabilities. In this work, we investigated the effects of m1A and m6A on both the structure and thermodynamic stability of a newly reported DNA minidumbbell formed by two TTTA tetranucleotide repeats. As revealed by the results of nuclear magnetic resonance spectroscopic studies, both m1A and m6A favored the formation of a T·m1A and T·m6A Hoogsteen base pair, respectively. More intriguingly, the m1A and m6A modifications brought about stabilization and destabilization effects on the DNA minidumbbell, respectively. This work provides new biophysical insights into the effects of adenine methylation on the structure and thermodynamic stability of DNA.

Development and characterization of Novel EST-based single-copy genic microsatellite DNA markers in white spruce and black spruce

Background Due mainly to large genome size and prevalence of repetitive sequences in the nuclear genome of spruce ( Picea ), it is very difficult to develop single-copy genomic microsatellite markers. We have developed and characterized 25 polymorphic, single-copy genic microsatellites from white spruce ( Picea glauca ) EST sequences and determined their informativeness in white spruce and black spruce ( Picea mariana ) and inheritance in black spruce. Methods and Results White spruce EST sequences from NCBI dbEST were searched for the presence of microsatellite repeats. Forty-seven sequences containing dinucleotide, trinucleotide, tetranucleotide and compound repeats were selected to develop primers. Twenty-five of the designed primer pairs yielded scorable amplicons, with single-locus patterns, and were characterized in 20 individuals each of white spruce and black spruce. All 25 microsatellites were polymorphic in white spruce and 24 in black spruce. The number of alleles at a locus ranged from 2 to 18, with a mean of 8.8 in white spruce, and from 1 to 17, with a mean of 7.6 in black spruce. The expected heterozygosity/polymorphic information content ranged from 0.10 to 0.92, with a mean of 0.67 in white spruce, and from 0 to 0.93, with a mean of 0.59 in black spruce. Conclusions Microsatellites with dinucleotide and compound repeats were more informative than those with trinucleotide and tetranucleotide repeats. Eighteen microsatellite markers polymorphic between the parents of a black spruce controlled cross inherited in a single-locus Mendelian fashion. The microsatellite markers developed can be applied for various genetics, genomics, breeding, and conservation studies and applications.

Instability of Non-Standard Microsatellites in Relation to Prognosis in Metastatic Colorectal Cancer Patients

Very few data are reported in the literature on the association between elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) and prognosis in advanced colorectal cancer. Moreover, there is no information available in relation to the response to antiangiogenic treatment. We analyzed EMAST and vascular endothelial growth factor-B (VEGF-B) microsatellite status, together with standard microsatellite instability (MSI), in relation to prognosis in 141 patients with metastatic colorectal cancer (mCRC) treated with chemotherapy (CT) alone (n = 51) or chemotherapy with bevacizumab (B) (CT + B; n = 90). High MSI (MSI-H) was detected in 3% of patients and was associated with progression-free survival (PFS; p = 0.005) and overall survival (OS; p < 0.0001). A total of 8% of cases showed EMAST instability, which was associated with worse PFS (p = 0.0006) and OS (p < 0.0001) in patients treated with CT + B. A total of 24.2% of patients showed VEGF-B instability associated with poorer outcome in (p = 0.005) in the CT arm. In conclusion, our analysis indicated that EMAST instability is associated with worse prognosis, particularly evident in patients receiving CT + B.

The Clinicopathological Features and Genetic Mutations in Gastric Cancer Patients According to EMAST and MSI Status

Background: There has been no report regarding the clinicopathological features and genetic mutations regarding elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in gastric cancer (GC). Methods: The correlation among EMAST status, microsatellite instability (MSI) status, mutations of common GC-related genes and 16 DNA repair-associated genes, and the clinicopathological features were analyzed. Results: Among the 360 GC patients enrolled, there were 76 (21.1%) with EMAST+ tumors and 284 with EMAST− tumors, and 59 (16.4%) were MSI-high (MSI-H) tumors, and 301 were microsatellite stable (MSS) tumors. Patients with EMAST+ tumors exhibited an earlier pathological T category and had more genetic mutations in the PI3K/AKT pathway, ARID1A and DNA repair-associated genes than those with EMAST− tumors. Patients with MSI-H tumors have more genetic mutations in the PI3K/AKT pathway and DNA repair-associated genes than those with MSS tumors. In the subgroup analysis for MSI-H GC, EMAST+ tumors were associated with earlier pathological T and N categories, earlier TNM stages, higher frequency of DNA-repair-associated genetic mutations, and a better survival rate than EMAST− tumors. Conclusions: PI3K/AKT pathway mutations may play an important role in EMAST+ and/or MSI-H GC. EMAST+/MSI-H tumors seem to represent a different subtype of gastric cancer from EMAST−/MSI-H tumors.

A Computational Method to Quantify the Effects of Slipped Strand Mispairing on Bacterial Tetranucleotide Repeats

The virulence and pathogenicity of bacterial pathogens are related to their adaptability to changing environments. One process enabling adaptation is based on minor changes in genome sequence, as small as a few base pairs, within segments of genome called simple sequence repeats (SSRs) that consist of multiple copies of a short sequence (from one to several nucleotides), repeated in series. SSRs are found in eukaryotes as well as prokaryotes, and length variation in them occurs at frequencies up to a million-fold higher than bacterial point mutations through the process of slipped strand mispairing (SSM) by DNA polymerase during replication. The characterization of SSR length by standard sequencing methods is complicated by the appearance of length variation introduced during the sequencing process that obscures the lower abundance repeat number variants in a population. Here we report a computational approach to correct for sequencing process-induced artifacts, validated for tetranucleotide repeats by use of synthetic constructs of fixed, known length. We apply this method to a laboratory culture of Histophilus somni, prepared from a single colony, and demonstrate that the culture consists of populations of distinct sequence phase and length variants at individual tetranucleotide SSR loci.