Novel breeding approach for Japanese flounder using atmosphere and room temperature plasma mutagenesis tool

Abstract

Background Artificial induction of mutagenesis is effective for genetic resource innovation and breeding. However, the traditional mutation methods for fish breeding are not convenient or safe for daily use. Hence, development of a simple, safe and effective mutagenesis method with a high mutation rate and applicability to multiple fish species, is needed. Results We reported the first successful mutagenesis in a marine aquaculture fish species, Japanese flounder, Paralichthys olivaceus , using a novel atmosphere and room temperature plasma (ARTP) mutagenesis tool. ARTP treatment time was optimized for the fertilized eggs and sperm, respectively. Eggs fertilized for 60 min were treated by ARTP with a radio-frequency power input of 120 W, and the ARTP treatment time was 25 min. Under an ARTP radio-frequency power input of 200 W, the optimal treatment time for sperm diluted with Ringer’s solution by 1:40 v /v was 10 min. The ARTP-treated group presented differences in morphological traits such as body height, total length among individuals at day 90 after hatching. Whole-genome sequencing was used to reveal the mutation features of ARTP-treated individuals collected at day 120 after hatching. In total, 69.25Gb clean data were obtained from three controls and eight randomly selected ARTP-treated individuals, revealing 240,722 to 322,978 SNPs and 82,149 to 86,798 InDels located in 17,394~18,457 and 12,907~13,333 genes, respectively. The average mutation rate reached 0.064% at the genome level. Gene ontology clustering indicated that genes associated with cell components, binding function, catalytic activity, cellular process, metabolic process and biological regulation processes had higher mutation rates. Conclusions ARTP mutagenesis is a useful method for breeding of fish species to accelerate the selection of economically important traits that would benefit the aquaculture industry, given the variety of mutations detected.

DNA Copy Number Variations as Markers of Mutagenic Impact

“…Analysis of the genome-wide structural consequences of chemical mutagen- or radiation-induced mutations in plants and animals obtained during selection works allows expanding understanding of mutagen-induced CNVs. In the reviews of breeding studies not only mutagen-induced CNVs but also indels in poplar [69] and fish [70] were included because of the few available examples, given that these genomic repeats differ only in size and can also characterize CNV mutagenesis.…”

Enhancing Activities of Salt-Tolerant Proteases Secreted by Aspergillus oryzae Using Atmospheric and Room-Temperature Plasma Mutagenesis

“…The activated chemical particles might change DNA fragments by extensive damage in the P–O bonds in phosphodiester, the C–N bond between ribose and base, as well as amino groups on the base . Previous investigations showed that ARTP was a more convenient, effective, and “greener” technology to generate a mutant library with sufficient diversity in comparison to the traditional ultraviolet and chemical mutagens. , The advantages of ARTP are attributed to its stronger DNA damage capacity in comparison to the traditional methods, leading to a higher mutation rate of microbe. So far, over 100 kinds of mutated microbes, such as fungi, bacteria, and microalgae, have been successfully obtained using ARTP technology, − but the application of ARTP in A.…”

Insights into the molecular-level effects of atmospheric and room-temperature plasma on mononucleotides and single-stranded homo- and hetero-oligonucleotides

“…However, by far we still hardly link the current deduced results to organism mutation mechanism by ARTP in vivo, since for in vivo mutation, in addition to the direct breakage of DNA at the first step, the complicated repair system for the damaged DNA during cell survival growth is another important factor, which make the mutation process analysis complicated. Recently, whole-genome sequencing also revealed the in vivo mutation features of Japanese flounder, a sea fish of Paralichthys olivaceus caused by ARTP mutagenesis, which showed multiple SNP and InDel mutation sites and the average mutation rate was up to 0.064% at the genome level, which was 2-3 order higher than the traditional mutagenesis methods 23 . According to bioinformatics analysis, the higher mutation rates were www.nature.com/scientificreports/ found in many key genes associated with cellular process, metabolic process, biological regulation processes, cell components, binding function and catalytic activity in mutated species of P. olivaceus 23 .…”

“…Recently, whole-genome sequencing also revealed the in vivo mutation features of Japanese flounder, a sea fish of Paralichthys olivaceus caused by ARTP mutagenesis, which showed multiple SNP and InDel mutation sites and the average mutation rate was up to 0.064% at the genome level, which was 2-3 order higher than the traditional mutagenesis methods 23 . According to bioinformatics analysis, the higher mutation rates were www.nature.com/scientificreports/ found in many key genes associated with cellular process, metabolic process, biological regulation processes, cell components, binding function and catalytic activity in mutated species of P. olivaceus 23 . By multiplex ARTP mutagenesis of Zymomonas mobilis, a bacterial strain for ethanol production, a mutant with improved tolerance to both high acetic acid concentration up to 8 g/L and low pH down to pH 3.5 was obtained, which showed enhanced growth and ethanol production under both sterilized/unsterilized conditions at pH 4.0 or 3.5 24 .…”

“…1). We have employed this system to mutate the genomes of various microbes and plants as well as animals, including bacteria, microalgae, fungi, and yeast 1,4,[19][20][21][22][23][24] . The results of those studies indicated that ARTP mutation is a rapid, effective, convenient, and multifaceted means of generating mutant libraries with sufficient diversity for the improvement of phenotypes.…”