Conversion of a normal maize hybrid into a waxy version using in vivo CRISPR/Cas9 targeted mutation activity

Waxy maize is a specialty maize that produces mainly amylopectin starch with special food or industrial values. The objective of this study was to overcome the limitations of wx mutant allele acquisition and breeding efficiency by conversion of parental lines from normal to waxy maize. The intended mutation activity was achieved by in vivo CRISPR/Cas9 machinery involving desired-target mutation of the Wx locus in the ZC01 background,abbreviated as ZC01-DTM^(wx). Triple selection was applied to segregants to obtain high genome background recovery with transgene-free wx mutations. The targeted mutation was identified, yielding six types of mutations among progeny crossed with ZC01-DTM^(wx).The amylopectin contents of the endosperm starch in mutant lines and hybrids averaged94.9%, while those of the wild-type controls were significantly(P < 0.01) lower, with an average of 76.9%. Double selection in transgene-free lines was applied using the Bar strip test and Cas9 PCR screening. The genome background recovery ratios of the lines were determined using genome-wide SNP data. That of lines used as male parents was as high as98.19% and that of lines used as female parents was as high as 86.78%. Conversion hybrids and both parental lines showed agronomic performance similar to that of their wild-type counterparts. This study provides a practical example of the efficient extension of CRISPR/Cas9 targeted mutation to industrial hybrids for transformation of a recalcitrant species.

Efficient generation of targeted point mutations in the Brassica oleracea var.botrytis genome via a modified CRISPR/Cas9 system

In this study,we used the modified CRISPR/Cas9 system to produce targeted point mutations in cauliflower.Acetolactate synthase(ALS)and Centromere-specific histone H3 variant(CENH3)genes were selected as the base-editing targets and hypocotyls of cauliflower were used as explants.For ALS gene,a C-to-T conversion in the Pro182 codon(CCT)can alter the encoded amino acid,likely resulting in herbicide resistance,and a C-to-T mutation in the Leu133 codon(CTT)in the CENH3 gene may produce a haploid inducer.Results indicated that the transformation efficiency was 1.8%–4.5%and the mutation efficiencies for the ALS and CENH3 genes were approximately 22%and 87%,respectively.The ALS mutant cauliflower showed strong herbicide resistance,with possible immediate implications for broadleaf weed control in cauliflower fields.

IVS8 ＋ 1 DelG, a Novel Splice Site Mutation Causing DFNA5 Deafness in a Chinese Family

Background： Nonsyndrornic hearing loss （NSHL） is highly heterogeneous, in which more than 90 causative genes have currently been identified. DFNA5 is one of the deathess genes that known to cause autosomal dominant NSHL. Until date, only five DFN.45 mutations have been described in eight families worldwide. In this study, we reported the identification of a novel pathogenic mutation causing DFNA5 deafness in a five-generation Chinese family. Methods： Alter detailed clinical evaluations of this family, the genomic DNA of three affected individuals was selected for targeted exome sequencing of 101 known deafness genes, as well as mitochondrial DNA and microRNA regions. Co-segregation analysis between the hearing loss and the candidate variant was confirmed in available family members by direct polymerase chain reaction （PCR）-Sanger sequencing. Real-time PCR （RT-PCR） was pertormed to investigate the potential effect of the pathogenic mutation on messenger RNA splicing. Results： Clinical evaluations revealed a similar deafness phenotype in this family to that of previously reported DFNA5 families with autosomal dominant, late-onset bearing loss. Molecular analysis identified a novel splice site mutation in DFNA5 intron 8 （IVSS＋ 1 delG）. The mutation segregated with the hearing loss of the family and was absent in 120 unrelated control DNA samples of Chinese origin. RT-PCR showed skipping of exon 8 in the mutant transcript. Conclusions： We identified a novel DFNA5 mutation IVS8＋1 delG in a Chinese family which led to skipping ofexon 8. This is the sixth DFNA5 mutation relates to hearing loss and the second one in DFNA5 intron 8. Our findings provide further support to the hypothesis that the DFNA5-associated hearing loss represents a mechanism of gain-of-function.

Highly efficient and genotype-independent barley gene editing based on anther culture

Recalcitrance to tissue culture and genetic transformation is the major bottleneck for gene manipulation in crops.In barley,immature embryos of Golden Promise have typically been used as explants for transformation.However,the genotype dependence of this approach limits the genetic modification of commercial varieties.Here,we developed an anther culture-based system that permits the effective creation of transgenic and gene-edited plants from commercial barley varieties.The protocol was tested in Golden Promise and four Australian varieties,which differed in phenology,callus induction,and green plant regeneration responses.Agrobacterium-mediated transformation was performed on microspore-derived callus to target the HvPDS gene,and T0 albinos with targeted mutations were successfully obtained from commercial varieties.Further editing of three targets was achieved with an average mutation rate of 53%in the five varieties.In 51 analyzed T0 individuals,Cas9 induced a large proportion(69%)of single-base indels and two-base deletions in the target sites,with variable mutation rates among targets and varieties.Both ontarget and off-target activities were detected in T1 progenies.Compared with immature embryo protocols,this genotype-independent platformcan deliver a high editing efficiency and more regenerant plants within a similar time frame.It shows promise for functional genomics and the application of CRISPR technologies for the precise improvement of commercial varieties.

Frequencies and mechanisms of pesticide resistance in Tetranychus urticae field populations in China

The two-spotted spider mite Tetranychus urticate is an important agricultural pest worldwide.It is extremely polyphagous and has developed resistance to many pesticides.Here,we assessed the pesticide resistance of seven field populations of T.urticae in China,their target site mutations and the activities of their detoxification enzymes.The results showed that abamectin and the traditional pesticides pyridaben,profenofos and bifenthrin had higher resistance or lower toxicity than more recently developed pesticides including chlorfenapyr,spinetoram,cyflumetofen,cyenopyrafen,bifenazate and B-azolemiteacrylic.The frequency of point mutations related to abamectin resistance,G314D in the glutamate-gated chloride channel 1(GluCl1)and G326E in GluC13,ranged 47%-70%and 0%-97%,respectively.The frequency of point mutations in A1215D and F1538I of the voltage-gated sodium channel gene(VGSC),which may increase resistance to pyrethroids,ranged 88%-100%and 10%-100%,respectively.For target sites related to organophosphate resistance,mutation frequencies ranged 25%-92%for G119S and 0%-23%for A201S in the acetycholinesterase gene(Ace).Mutation G126S in the bifenazate resistance-related cytochrome b gene(Cytb)was observed in three of the seven T.urticae populations.Higher activities of detoxification enzymes(P450,GST,CarEs and UGTs)were observed in two T.urticae populations,with significant difference in the XY-SX population.These results provide useful information on the status of pesticide resistance of T.urticae in China and suggest that T.urticae field populations may have multiple resistance mechanisms.