Generation of marker‐free transgenic hexaploid wheat via an <i>Agrobacterium</i>‐mediated co‐transformation strategy in commercial Chinese wheat varieties

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Summary Kernel size is an important trait determining cereal yields. In this study, we cloned and characterized TaDA1, a conserved negative regulator of kernel size in wheat (Triticum aestivum). The overexpression of TaDA1 decreased the size and weight of wheat kernels, while its down‐regulation using RNA interference (RNAi) had the opposite effect. Three TaDA1‐A haplotypes were identified in Chinese wheat core collections, and a haplotype association analysis showed that TaDA1‐A‐HapI was significantly correlated with the production of larger kernels and higher kernel weights in modern Chinese cultivars. The haplotype effect resulted from a difference in TaDA1‐A expression levels between genotypes, with TaDA1‐A‐HapI resulting in lower TaDA1‐A expression levels. This favourable haplotype was found having been positively selected during wheat breeding over the last century. Furthermore, we demonstrated that TaDA1‐A physically interacts with TaGW2‐B. The additive effects of TaDA1‐A and TaGW2‐B on kernel weight were confirmed not only by the phenotypic enhancement arising from the simultaneous down‐regulation of TaDA1 and TaGW2 expression, but also by the combinational haplotype effects estimated from multi‐environment field data from 348 wheat cultivars. A comparative proteome analysis of developing transgenic and wild‐type grains indicated that TaDA1 and TaGW2 are involved in partially overlapping but relatively independent protein regulatory networks. Thus, we have identified an important gene controlling kernel size in wheat and determined its interaction with other genes regulating kernel weight, which could have beneficial applications in wheat breeding.

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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TaDA1, a conserved negative regulator of kernel size, has an additive effect with TaGW2 in common wheat (Triticum aestivum L.)

Hao

et al. 2019

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“…Paired‐end (2 × 250) sequencing was conducted with a total of 500 cycles. Sequence analysis was conducted with CRISPR‐DAV (Wang et al ., ) to discover the modification.…”

Section: Methodsmentioning

confidence: 99%

Development of an Agrobacterium‐delivered CRISPR/Cas9 system for wheat genome editing

Zhang

Hua

Gupta

et al. 2019

163

104

Summary CRISPR /Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR /Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR /Cas9 complex. Due to the high frequency of gene silencing associated with co‐transferred plasmid backbone and low edit rate in wheat, a large T 0 transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium ‐delivered CRISPR /Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR /Cas9 system, we have developed 68 edit mutants for four grain‐regulatory genes, Ta CKX 2‐1 , Ta GLW 7 , Ta GW 2, and Ta GW 8 , in T 0 , T 1 , and T 2 generation plants at an average edit rate of 10% without detecting off‐target mutations in the most Cas9‐active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160‐bp deletion in Ta CKX 2‐D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium ‐delivered CRISPR /Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR /Cas9 remains active for novel mutations through generations.

“…In TSI events where the GOI is under the control of the 35S promoter that also drives the expression of the cry2Ae gene from the original cotton event, the double presence of the 35S promoter might have influenced the level of GOI expression in some events. Especially since it has been reported that the 35S sequence is known to be susceptible to silencing by methylation (Okumura et al ., ; Wang et al ., ). However, we believe that another, more general trigger is at play since we observed silencing in TSI events with other GOI promoters while the expression of the 35S driven cry2Ae gene was stable and not affected in any of the plants tested.…”

Section: Discussionmentioning

confidence: 97%

Impact of differential DNA methylation on transgene expression in cotton (Gossypium hirsutum L.) events generated by targeted sequence insertion

Verkest¹,

Bourout²,

Debaveye³

et al. 2019

Summary Targeted Genome Optimization ( TGO ) using site‐specific nucleases to introduce a DNA double‐strand break ( DSB ) at a specific target locus has broadened the options available to breeders for generation and combination of multiple traits. The use of targeted DNA cleavage in combination with homologous recombination ( HR )‐mediated repair, enabled the precise targeted insertion of additional trait genes (2m epsps, hppd, axmi115 ) at a pre‐existing transgenic locus in cotton. Here we describe the expression and epigenome analyses of cotton Targeted Sequence Insertion ( TSI ) events over generations. In a subset of events, we observed variability in the level of transgene ( hppd , axmi115 ) expression between independent but genetically identical TSI events. Transgene expression could also be differential within single events and variable over generations. This expression variability and silencing occurred independently of the transgene sequence and could be attributed to DNA methylation that was further linked to different DNA methylation mechanisms. The trigger(s) of transgene DNA methylation remains elusive but we hypothesize that targeted DSB induction and repair could be a potential trigger for DNA methylation.