Expanding the Benefits of Tnt1 for the Identification of Dominant Mutations in Polyploid Crops: A Single Allelic Mutation in the MsNAC39 Gene Produces Multifoliated Alfalfa

Most major crops are polyploid species and the production of genetically engineered cultivars normally requires the introgression of transgenic or gene-edited traits into elite germplasm. Thus, a main goal of plant research is the search of systems to identify dominant mutations. In this article, we show that the Tnt1 element can be used to identify dominant mutations in allogamous tetraploid cultivated alfalfa. Specifically, we show that a single allelic mutation in the MsNAC39 gene produces multifoliate leaves (mfl) alfalfa plants, a pivot trait of breeding programs of this forage species. Finally, we discuss the potential application of a combination of preliminary screening of beneficial dominant mutants using Tnt1 mutant libraries and genome editing via the CRISPR/Cas9 system to identify target genes and to rapidly improve both autogamous and allogamous polyploid crops.

A novel recessive PDZD7 bi-allelic mutation in an Iranian family with non-syndromic hearing loss

Background Autosomal recessive non-syndromic hearing loss (ARNSHL) is genetically and phenotypically heterogeneous with over 110 genes causally implicated in syndromic and non-syndromic hearing loss. Here, we investigate the genetic etiology of deafness in two GJB2 and GJB6 negative patients presenting with pre-lingual, progressive, severe hearing loss. Methods Targeted exome sequencing (TES) using Next Generation Illumina Sequencing was used to analyze the exonic and some other important genomic regions of 154 genes in the proband. Subsequently, the mutation found was confirmed by Sanger sequencing in other affected sibling and healthy family members. The possible impact of the reported mutation on the corresponding protein was also evaluated by using bioinformatics tools. Moreover, the affected patients underwent audiological and ophthalmic evaluations. Results TES identified a novel homozygous missense mutation c.251T>C (p.I84T) in exon 3 of PDZD7 gene. In addition, segregation and phenotype-genotype correlation analysis as well as in-silico evaluations confirmed the autosomal recessive inheritance pattern and disease-causing nature of mutation found. Conclusions In overall, our finding could expand the pathogenic mutations spectrum and strengthens the clinical importance of the PDZD7 gene in ARNSHL patients. It can also aid to conduct genetic counseling, prenatal diagnosis and clinical management of these types of genetic disorders.

Comparison of the effects of introducing the CRISPR/Cas9 system by microinjection and electroporation into porcine embryos at different stages

Objective Cytoplasmic microinjection and electroporation of the CRISPR/Cas9 system into zygotes are used for generating genetically modified pigs. However, these methods create mosaic mutations in embryos. In this study, we evaluated whether the gene editing method and embryonic stage for gene editing affect the gene editing efficiency of porcine embryos. Results First, we designed five guide RNAs (gRNAs) targeting the B4GALNT2 gene and evaluated mutation efficiency by introducing each gRNA with Cas9 protein into zygotes by electroporation. Next, the optimized gRNA with Cas9 protein was introduced into 1-cell and 2-cell stage embryos by either microinjection or electroporation. The sequence of gRNA affected the bi-allelic mutation rate and mutation efficiency of blastocysts derived from electroporated embryos. Microinjection significantly decreased the cleavage rates in each embryonic stage and blastocyst formation rates in 2-cell stage embryos compared with electroporation (p < 0.05). However, the bi-allelic mutation rate and mutation efficiency of blastocysts from the 1-cell stage embryos edited using microinjection were significantly higher (p < 0.05) than those of blastocysts from the 2-cell stage embryos edited by both methods. These results indicate that the gene editing method and embryonic stage for gene editing may affect the genotype and mutation efficiency of the resulting embryos.

A Novel Recessive PDZD7 Bi-Allelic Mutation in an Iranian Family with Non-Syndromic Hearing Loss

BackgroundAutosomal recessive non-syndromic hearing loss (ARNSHL) is genetically and phenotypically heterogeneous with over 110 genes causally implicated in syndromic and non-syndromic hearing loss (HL). Here, we investigate the genetic etiology of deafness in twoGJB2 and GJB6 negative patients presenting with pre-lingual, progressive, severe hearing loss.MethodsTargeted exome sequencing (TES) using Next Generation Illumina Sequencing was used to analyze the exonic and some other important genomic regions of 154 genes in the proband. Subsequently, the mutation found was confirmed by Sanger sequencing in other affected sibling and healthy family members. The possible impact of the reported mutation on the corresponding protein was also evaluated by using bioinformatics tools. Moreover, the affected patients underwent audiological and ophthalmic evaluations.ResultsTES identified a novel homozygous missense mutation c.251T>C (p.I84T) in exon 3 of PDZD7 gene. In addition, segregation and phenotype-genotype correlation analysis as well as in-silico evaluations confirmed the autosomal recessive inheritance pattern and disease-causing nature of mutation found. ConclusionsIn overall, our finding could expand the pathogenic mutations spectrum and strengthens the clinical importance of the PDZD7 gene in ARNSHL patients. It can also aid to conduct genetic counseling, prenatal diagnosis and clinical management of these types of genetic disorders.

Antibiotic induced biofilm formation of novel multidrug resistant Acinetobacter baumannii ST2121 clone

The aim of this study was to identify antimicrobial resistance and virulence factor genes exhibited by multidrug resistant (MDR) Acinetobacter baumannii, to analyze biofilm formation and to investigate clonal subtypes of isolate. Whole genome sequencing was done by Illumina NovaSeq 6,000 platform and multilocus sequence typing (MLST) was performed by Oxford and Pasteur typing schemes. Influence of imipenem and levofloxacin on biofilm formation was investigated in 96-well plates at 3 replicates. The strain was found to carry OXA-23, OXA-51-like, AmpC and TEM-1 beta-lactamases. The sequence of the blaOXA-51-like gene has been identified as a blaOXA-66. According to Pasteur MLST scheme the strain displayed ST2 allelic profile. However, based on Oxford MLST scheme this strain represents the new ST2121, as the gdhB gene has a single allelic mutation namely, the gdhB-227. It was determined that MDR isolate carried bap, basABCDFGHIJ, csuA/BABCDE, bauABCDEF, plcD, pgaABCD, entE, barAB, ompA, abaIR, piT2EAFTE/AUBl, fimADT, cvaC, bfmR, bfmS virulence genes. In our study imipenem induced the highest biofilm formation at a concentration of 32 µg/ml and levofloxacin at a concentration of 16 µg/ml. In conclusion, we detected a new MDR A. baumannii ST2121 clone harboring blaOXA-66 gene that has been reported for the first time in Turkey.

Genotypic and allelic frequencies of MDR1 gene in dogs in Italy

BackgroundA mutation in the canine multidrug resistance MDR1 gene (also referred as ABCB1), encoding for the multidrug resistance (MDR) P-glycoprotein (P-gp) transponder, causes a pathological condition known as ‘ivermectin toxicosis’. The causative mutation, known since 2001, has been described to affects sheep herding breeds related to collie lineage. The present study is a retrospective investigation of the presence of MDR1 mutated allele in Italian dog populations in a 5 years’ time lapse. The aim of the research is to offer a deep knowledge in MDR1 allelic and genotypic frequencies in canine breeds and populations raised in Italy.MethodsGenotype data for the 4-bp deletion (c296_299del4) in MDR1 gene from 811 dogs belonging to 32 breeds/populations were collected.ResultsThe mutated allele has been found in 9 out of 31 breeds: Rough Collie, Smooth Collie, Border Collie, Bearded Collie, Shetland Sheepdog, Australian Shepherd, White Swiss Shepherd, Old English Sheepdog, Whippet and also in crossbreed. The breeds with the highest allelic mutation frequency are Smooth and Rough Collies with 75 per cent and 66 per cent of mutant MDR1 allele, respectively.ConclusionsThe results support the usefulness of this genetic analysis to optimise medical care in dogs at risk of multidrug resistance and to create an objective basis in breeding programme definition and in the risk evaluation in different breeds.