Restriction Enzyme Double Digestion v1

Enzyme digestion is to cut the DNA molecule and the carrier molecule at the sticky end to obtain the corresponding sticky end connection.

De Novo SNP Discovery and Genotyping of Iranian Pimpinella Species Using ddRAD Sequencing

The species of Pimpinella, one of the largest genera of the family Apiaceae, are traditionally cultivated for medicinal purposes. In this study, high-throughput double digest restriction-site associated DNA sequencing technology (ddRAD-seq) was used to identify single nucleotide polymorphisms (SNPs) in eight Pimpinella species from Iran. After double-digestion with the enzymes HpyCH4IV and HinfI, a total of 334,702,966 paired-end reads were de novo assembled into 1,270,791 loci with an average of 28.8 reads per locus. After stringent filtering, 2440 high-quality SNPs were identified for downstream analysis. Analysis of genetic relationships and population structure, based on these retained SNPs, indicated the presence of three major groups. Gene ontology and pathway analysis were determined by using comparison SNP-associated flanking sequences with a public non-redundant database. Due to the lack of genomic resources in this genus, our present study is the first report to provide high-quality SNPs in Pimpinella based on a de novo analysis pipeline using ddRAD-seq. This data will enhance the molecular knowledge of the genus Pimpinella and will provide an important source of information for breeders and the research community to enhance breeding programs and support the management of Pimpinella genomic resources.

An approach using ddRADseq and machine learning for understanding speciation in Antarctic Antarctophilinidae gastropods

Sampling impediments and paucity of suitable material for molecular analyses have precluded the study of speciation and radiation of deep-sea species in Antarctica. We analyzed barcodes together with genome-wide single nucleotide polymorphisms obtained from double digestion restriction site-associated DNA sequencing (ddRADseq) for species in the family Antarctophilinidae. We also reevaluated the fossil record associated with this taxon to provide further insights into the origin of the group. Novel approaches to identify distinctive genetic lineages, including unsupervised machine learning variational autoencoder plots, were used to establish species hypothesis frameworks. In this sense, three undescribed species and a complex of cryptic species were identified, suggesting allopatric speciation connected to geographic or bathymetric isolation. We further observed that the shallow waters around the Scotia Arc and on the continental shelf in the Weddell Sea present high endemism and diversity. In contrast, likely due to the glacial pressure during the Cenozoic, a deep-sea group with fewer species emerged expanding over great areas in the South-Atlantic Antarctic Ridge. Our study agrees on how diachronic paleoclimatic and current environmental factors shaped Antarctic communities both at the shallow and deep-sea levels, promoting Antarctica as the center of origin for numerous taxa such as gastropod mollusks.

Reconstruction and optimized expression of a synthetic secretory leukocyte protease inhibitor (SLPI) gene in Escherichia coli BL21

An active substance that has the greatest effect on wound healing is Secretory Leukocyte ProteaseInhibitor (SLPI). It is known that the SLPI encoding genes can be isolated and expressed onamnion membrane. Previous studies, we isolated and optimized the SLPI gene throughEscherichia coli BL21 (DE3) mediated pET101/DTOPO, which expressed active recombinanthuman SLPI (rhSLPI ) stored in pET-ESLPI. However, the expression of the rhSLPI products hasnot yet been accomplished. In this study, we optimized SLPI expression by developing a syntheticSLPI gene based on amino acid sequences with codons and expressed in E. coli BL21 to give themaximum expression. We used pUC57 and pET-32a plasmids to promote the cloning of syntheticSLPI genes. A codon-optimized SLPI gene was successfully synthesized with codon adaptationindex value showing the distribution of codon usage frequency along the length of the genesequence. In addition, the pET-SLPIopt fusion protein was successfully optimized with band sizesof 5900bp (pET-32a) and 413bp (SLPI) by double-digestion of NcoI and EcoI restriction enzymes.After the pET-SLPIopt was induced with various IPTG concentrations (50, 100 and 500 uM) at30°C, both soluble and insoluble fractions were analyzed as a result of SDS-PAGE which showedthat the fusion protein, expressed predominantly in the supernatant, was 29.18 kDa. Our reportedfindings the recombinant protein of SLPI through pET-32a plasmid could be expressed indissolved form.

Deciphering the unique SNPs among leading Indian tomato cultivars using double Digestion Restriction Associated DNA sequencing

World-wide grown and consumed tomato (Solanum lycopersicum) is used as model crop for newcultivar and fruit development. Genetic and genomic studieson Indian tomato cultivars will provide an insight that will enable development of breeding strategies and crop improvement. The present study aims to identifythe high quality common and unique SNPs and INDELs, present in 9 different Indian tomato cultivars using double digest restriction site-associated DNA sequencing (ddRAD-seq). A total of 36.8 million raw reads were generated for selected cultivars and an average of 94% high-quality reads of each were uniquely aligned to the reference tomato genome (SLv3.0). Out of 6,957 SNPs and188 INDELs, we found 1,165 SNPs and 68 INDELs in genic regions. The genetic relationship among these cultivars suggested 4well-differentiated groups of cultivars. Similarly, 7 and 33 SNPs were identified in chloroplast and mitochondrial genomes of tomato. SNPs markers were identified for common and specific genes associated with different pathways and their gene ontology (GO) annotated. These SNPs/INDELs could be useful as markers for variety identification for genetic purity analysis. Findings from this work will be useful to the research community, particularly, plant breeders, as a resource for SNP marker development.

Identification and Mapping of Eastern Filbert Blight Resistance Quantitative Trait Loci in European Hazelnut Using Double Digestion Restriction Site Associated DNA Sequencing

European hazelnut (Corylus avellana L.) is an economically important edible nut producing species, which ranked sixth in world tree nut production in 2016. European hazelnut production in the United States is primarily limited to the Willamette Valley of Oregon, and currently nonexistent in the eastern United States because of the presence of a devastating endemic disease, eastern filbert blight (EFB) caused by Anisogramma anomala (Peck) E. Muller. The primary commercial means of control of EFB to date is through the development and planting of genetically resistant european hazelnut cultivars, with an R-gene introduced from the obsolete, late-shedding pollinizer ‘Gasaway’. Although the ‘Gasaway’ resistance source provides protection against EFB in the Pacific northwestern United States (PNW), recent reports have shown that it is not effective in parts of the eastern United States. This may be in part because the identification and selection of ‘Gasaway’ and ‘Gasaway’-derived cultivars occurred in an environment (PNW) with limited genetic diversity of A. anomala. The objectives of the current research were to develop a genetic linkage map using double digestion restriction site associated DNA sequencing (ddRADseq) and identify quantitative trait loci (QTL) markers associated with EFB resistance from the resistant selection Rutgers H3R07P25 from southern Russia. A mapping population composed of 119 seedling trees was evaluated in a geographic location (New Jersey) where the EFB fungus is endemic, exhibits high disease pressure, and has a high level of genetic diversity. The completed genetic linkage map included a total of 2217 markers and spanned a total genetic distance of 1383.4 cM, with an average marker spacing of 0.65 cM. A single QTL region associated with EFB resistance from H3R07P25 was located on european hazelnut linkage group (LG) 2 and was responsible for 72.8% of the phenotypic variation observed in the study. Based on its LG placement, origin, and disease response in the field, this resistance source is different from the ‘Gasaway’ source located on LG6. The current results, in combination with results from previous research, indicate that the H3R07P25 source is likely exhibiting resistance to a broader range of naturally occurring A. anomala isolates. As such, H3R07P25 will be important for the development of new european hazelnut germplasm that combines EFB resistance from multiple sources in a gene pyramiding approach. Identification of EFB resistance in high disease pressure environments representing a diversity of A. anomala populations is likely a requirement for identifying plants expressing durable EFB resistance, which is a precursor to the development of a commercially viable european hazelnut industry in the eastern United States.