Abstract
Quickly and precisely gain genetically enhanced breeding elites with value-adding performance traits is desired by the crop breeders all the time. The present of gene editing technologies, especially the CRISPR/Cas9 system with the capacities of efficiency, versatility and multiplexing provides a reasonable expectation towards breeding goals. For exploiting possible application to accelerate the speed of process at breeding by CRISPR/Cas9 technology, in this study, the Agrobacterium tumefaciens-mediated CRISPR/Cas9 system transformation method was used for obtaining tomato ALC gene mutagenesis and replacement, in absence and presence of the homologous repair template. The average mutation frequency (72.73%) and low replacement efficiency (7.69%) were achieved in T0 transgenic plants respectively. None of homozygous mutation was detected in T0 transgenic plants, but one plant carry the heterozygous genes (Cas9/*-ALC/alc) was stably transmitted to T1 generations for segregation and genotyping. Finally, the desired alc homozygous mutants without T-DNA insertion (*/*-alc/alc) in T1 generations were acquired and further confirmed by genotype and phenotype characterization, with highlight of excellent storage performance, thus the recessive homozygous breeding elites with the character of long-shelf life were generated. Our results support that CRISPR/Cas9-induced gene replacement via HDR provides a valuable method for breeding elite innovation in tomato.
Highlights
The long-shelf life is a critical trait for the quality of fleshy fruit, and it is one of the major objectives in breeding programs as it influences fruit marketability for both farmer’s and consumer’s perception
The results show that both pre-determined locus mutagenesis and targeted gene replacement events can be occurred respectively, and the allele of ALC was replaced by alc gene via HDR repair pathway
To detect the Cas[9] endonuclease with a sgRNA can generate DSBs and alter ALC gene sequences through error-prone NHEJ repair, a codon optimized version of Cas[9] is controlled by a CaMV 35S promoter, the sgRNA is expressed by the AtU6 promoter (Supplementary Fig. 1), and both elements are constructed to the same T-DNA vector of pCAM1301, the sgRNA was designed with the guide sequence matching a 20-bp region alongside the protospacer-adjacent motif sequence (PAM) within the ALC gene (Fig. 1A)
Summary
The long-shelf life is a critical trait for the quality of fleshy fruit, and it is one of the major objectives in breeding programs as it influences fruit marketability for both farmer’s and consumer’s perception. The molecular basis of the alc mutation is the replacement of thymine by adenine in position 317 of the coding sequence of the NOR gene, this one base pair mutation is a nonsynonymous amino acid change, namely the valin (Val) was replaced by the aspartic acid (Asp). Confirming it is an allele of nor gene[8]. The date presents here hold great potential for tomato genetics and breeding
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