Optimization of Genotyping-by-sequencing (GBS) in Chrysanthemums: Selecting Proper Restriction Enzymes for GBS Library Construction

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Nan Wang ◽  
Yibing Yuan ◽  
Hui Wang ◽  
Diansi Yu ◽  
Yubo Liu ◽  
...  

Abstract Genotyping-by-Sequencing (GBS) is a low-cost, high-throughput genotyping method that relies on restriction enzymes to reduce genome complexity. GBS is being widely used for various genetic and breeding applications. In the present study, 2240 individuals from eight maize populations, including two association populations (AM), backcross first generation (BC1), BC1F2, F2, double haploid (DH), intermated B73 × Mo17 (IBM), and a recombinant inbred line (RIL) population, were genotyped using GBS. A total of 955,120 of raw data for SNPs was obtained for each individual, with an average genotyping error of 0.70%. The rate of missing genotypic data for these SNPs was related to the level of multiplex sequencing: ~ 25% missing data for 96-plex and ~ 55% for 384-plex. Imputation can greatly reduce the rate of missing genotypes to 12.65% and 3.72% for AM populations and bi-parental populations, respectively, although it increases total genotyping error. For analysis of genetic diversity and linkage mapping, unimputed data with a low rate of genotyping error is beneficial, whereas, for association mapping, imputed data would result in higher marker density and would improve map resolution. Because imputation does not influence the prediction accuracy, both unimputed and imputed data can be used for genomic prediction. In summary, GBS is a versatile and efficient SNP discovery approach for homozygous materials and can be effectively applied for various purposes in maize genetics and breeding.


2019 ◽  
Vol 97 (3) ◽  
pp. 381
Author(s):  
Jorge Ricaño-Rodríguez ◽  
Enrique Hipólito-Romero ◽  
José M. Ramos-Prado ◽  
Eliezer Cocoletzi-Vásquez

<p><strong>Background:</strong> Single nucleotide polymorphisms (SNPs) have been identified in <em>Theobroma cacao</em> through a genotyping-by-sequencing approach. Through this research it is shared for the first time a set of results related to genetic variability and nature of conserved coding regions of reduced nucleotide sequences of mexican native varieties of cocoa.</p><p><strong>Hypothesis:</strong> Obtaining reduced genomes of <em>T. cacao</em> specimens by restriction enzymes (REs) allows the characterization of single nucleotide polymorphisms (SNPs) as well as conserved coding regions (CDs).</p><p><strong>Species of study and dates:</strong> <em>Theobroma cacao </em>L. (Malvaceae)</p><p>Study site: <em>Theobroma cacao</em> twigs came from traditional agroforestry plots located in the municipalities of Cardenas, Huimanguillo, Comalcalco, Paraiso, Jalpa de Mendez and Cunduacan, Tabasco, as well as Ixtacomitan and Pichucalco, Chiapas, Mexico; and they were collected and grafted among May and June from 2018.</p><p><strong>Methods:</strong> A method of genotyping-by-sequencing for the characterization of biobanks was developed. Filtering of crude sequences, genomic assembly, identification of SNPs, taxonomic molecular characterization and characterization of coding regions as well as minimum evolution of protein transcripts were performed.</p><p><strong>Results:</strong> <em>Theobroma cacao</em> samples showed different SNPs percentages (2 – 11 %) and the molecular evolution analyzes suggested similar maximum compound probabilities respect to their phylogeny. Conserved sequences were observed in the genomes´ coding regions, which suggest heuristic ontological predictions that have been evolutionarily regrouped in five clusters related to transcription processes and secondary metabolism.</p><strong>Conclusions:</strong> The GBS method allows to identify SNPs in cocoa. The characterization of reduced genomes determined the structural and transcriptional correlation between the samples and the reference genome of cacao Criollo.


2019 ◽  
Vol 124 (4) ◽  
pp. 543-551 ◽  
Author(s):  
Davide Scaglione ◽  
Sara Pinosio ◽  
Fabio Marroni ◽  
Eleonora Di Centa ◽  
Alice Fornasiero ◽  
...  

Abstract Background and Aims The advent of molecular breeding is advocated to improve the productivity and sustainability of second-generation bioenergy crops. Advanced molecular breeding in bioenergy crops relies on the ability to massively sample the genetic diversity. Genotyping-by-sequencing has become a widely adopted method for cost-effective genotyping. It basically requires no initial investment for design as compared with array-based platforms which have been shown to offer very robust assays. The latter, however, has the drawback of being limited to analyse only the genetic diversity accounted during selection of a set of polymorphisms and design of the assay. In contrast, genotyping-by-sequencing with random sampling of genomic loci via restriction enzymes or random priming has been shown to be fast and convenient but lacks the ability to target specific regions of the genome and to maintain high reproducibility across laboratories. Methods Here we present a first adoption of single-primer enrichment technology (SPET) which provides a highly efficient and scalable system to obtain targeted sequence-based large genotyping data sets, bridging the gaps between array-based systems and traditional sequencing-based protocols. To fully explore SPET performance, we conducted a benchmark study in ten Zea mays lines and a large-scale study of a natural black poplar population of 540 individuals with the aim of discovering polymorphisms associated with biomass-related traits. Key Results Our results showed the ability of this technology to provide dense genotype information on a customized panel of selected polymorphisms, while yielding hundreds of thousands of untargeted variable sites. This provided an ideal resource for association analysis of natural populations harbouring unexplored allelic diversities and structure such as in black poplar. Conclusion The improvement of sequencing throughput and the development of efficient library preparation methods has made it feasible to carry out targeted genotyping-by-sequencing experiments cost-competitively with either random complexity reduction systems or traditional array-based platforms, while maintaining the key advantages of both technologies.


Author(s):  
Habtamu Ayalew ◽  
Joshua D Anderson ◽  
Nick Krom ◽  
Yuhong Tang ◽  
Twain J Butler ◽  
...  

Abstract Triticale, a hybrid species between wheat and rye, is one of the newest additions to the plant kingdom with a very short history of improvement. It has very limited genomic resources because of its large and complex genome. Objectives of this study were to generate dense marker data, understand genetic diversity, population structure, linkage disequilibrium (LD), and estimate accuracies of commonly used genomic selection (GS) models on forage yield of triticale. Genotyping-by-sequencing (GBS), using PstI and MspI restriction enzymes for reducing genome complexity, was performed on a triticale diversity panel (n = 289). After filtering for biallelic loci with more than 70% genome coverage, and minor allele frequency (MAF) &gt; 0.05, de novo variant calling identified 16,378 single nucleotide polymorphism (SNP) markers. Sequences of these variants were mapped to wheat and rye reference genomes to infer their homologous groups and chromosome positions. About 45% (7,430), and 58% (9,500) of the de novo identified SNPs were mapped to the wheat and rye reference genomes, respectively. Interestingly, 28.9% (2,151) of the 7,430 SNPs were mapped to the D genome of hexaploid wheat, indicating substantial substitution of the R genome with D genome in cultivated triticale. About 27% of marker pairs were in significant LD with an average r2 &gt; 0.18 (P &lt; 0.05). Genome-wide LD declined rapidly to r2 &lt; 0.1 beyond 10 kb physical distance. The three sub-genomes (A, B, and R) showed comparable LD decay patterns. Genetic diversity and population structure analyses identified five distinct clusters. Genotype grouping did not follow prior winter vs. spring type classification. However, one of the clusters was largely dominated by winter triticale. Genomic selection accuracies were estimated for forage yield using three commonly used models with different training population sizes and marker densities. Genomic selection accuracy increased with increasing training population size while gain in accuracy tended to plateau with marker densities of 2,000 SNPs or more. Average GS accuracy was about 0.52, indicating the potential of using GS in triticale forage yield improvement.


Author(s):  
Tamkeen Fatima ◽  
Farah Zeb ◽  
A. Dar Farooq

Background: CYP2D6 is to be considered the most pronounced gene in pharmacegenetic field which is involved in metabolizing ~25% of all clinically used neuroleptic drugs and other antidepressants. We designed a study to evaluate differential expression of CYP2D6*4 and CYP2D6*10 variants which are very prevalent in Asian countries and exhibit variation in drug metabolizing ability that affect therapeutic responses. Objective: The purpose of this study is to determine the genotypic frequencies of CYP2D6 *1 (normal metabolizer), *4 (poor metabolizer) and *10 (intermediate metabolizer) variants among schizophrenic subjects and compared with control group from a sub-set of Karachi population. Method: Genomic deoxyribonucleic acid (DNA ) was extracted and amplified with CYP2D6*4 and *10 primers using polymerase chain reaction (PCR) and digested by Bacillus stereothermophilus (BstN1) and Hemophilus parahemolyticus (Hph1) restriction enzymes. The digested bands were identified as wild type or mutants and their genotypic frequencies were estimated statistically by Hardy-Weinberg equation (HWE) and analyzed further under non-parametric Chi-square test. Results: The results mentioned the frequencies of CYP2D6*1 wild allele (57%) which produces functional enzyme in normal subjects but CYP2D6*4 variant (9%) that produces non-functional enzyme and CYP2D6*10 allele (70%) produces altered enzyme with reduced activity that was most prevalent in schizophrenic patients. Conclusion : Genotyping of CYP2D6 alleles among schizophrenic patients indicated prevalence of *4 and *10 variants in Karachi population producing non-functional and reduced functional drugs metabolizing enzymes respectively that increases the incurability rate of schizophrenia. Therefore, CYP2D6 gene screening program should be conducted routinely in clinical practice to help clinicians to prescribing appropriate doses according to patient’s genotype and minimize the sufferings of schizophrenia. Discussion: In last, drug response is a complex phenomenon that is dependent on genetic and environmental factors. CYP2D6 polymorphism may un-cured the schizophrenia due to improper drug metabolism and protein-proteins interaction that may alter the antipsychotic drugs metabolism among patients with variable drug resposes. Gene testing system need to establish for analyzing maximum patient’s genotypes predicted with poor metabolizer, intermediate metabolizer and ultrarapid metabolizer for the adjustment of antipsychotic drugs.


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