RNAi-mediated downregulation of AcCENH3 can induce in vivo haploids in onion (Allium cepa L.).

Haploid induction (HI) holds great promise in expediting the breeding process in onion, a biennial cross-pollinated crop. We used the CENH3-based genome elimination technique in producing a HI line in onion. Here, we downregulated AcCENH3 using the RNAi approach without complementation in five independent lines. Out of five events, only three could produce seeds upon selfing. The progenies showed poor seed set and segregation distortion, and we were unable to recover homozygous knockdown lines. The knockdown lines showed a decrease in accumulation of AcCENH3 transcript and protein in leaf tissue. The decrease in protein content in transgenic plants was correlated with poor seed set. When the heterozygous knockdown lines were crossed with wild-type plants, progenies showed HI by genome elimination of the parental chromosomes from AcCENH3 knockdown lines. The HI efficiency observed was between 0 and 4.63% in the three events, and it was the highest (4.63%) when E1 line was crossed with wildtype. Given the importance of doubled haploids in breeding programmes, the findings from our study are poised to significantly impact onion breeding.

Development of modified CTAB and Trizol protocols to isolate high molecular weight (HMW) RNA from polyphenol and polysaccharides rich pigeonpea (Cajanuscajan (L.) Millsp.

Pigeonpea (Cajanuscajan L.) is a legume crop that contains high levels of polyphenolic compounds and polysaccharides that become a hindrance in extracting good-quality and enough amount of RNA from its tissues. With the existing methods of RNA isolation, the phenolic compounds may co-precipitate or bind to the RNA giving false results. Therefore, in the present study, we have modified conventional CTAB and Trizol-based methods which resulted in good quality with the absorbance A260/A280 ratios in the range of 1.83 to 1.98 and A260/230 ratios in the range of 2.0-2.23, revealed RNA to be of high purity and free of contaminants. Both of the proposed protocols yielded a good quantity of RNA ranging from 289 to 422µg per gram of tissue. Distinctly visible bands of 28S and 18S rRNA were observed without degradation or smear, which indicated the presence of intact RNA. RT-PCR analysis showed that isolated RNA was quantitatively sufficient and compliant for the subsequent gene expression analysis.

CRISPR/Cas9-mediated editing of PHYTOENE DESATURASE gene in onion (Allium cepa L.).

Introduction: Clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) is a precise genome editing tool used to introduce genetic modifications in a wide range of crop species. Thus far, there is no report of CRISPR/Cas9-mediated genome editing in onions (Allium cepa L.). Methods: In the present study, we targeted two exons of the gene coding for Phytoene desaturase (AcPDS) in onion cv. Bhima Super. The sgRNA-carrying constructs were co-cultivated with 8-week-old embryogenic calli using an Agrobacterium-mediated transformation protocol and incubated on the media without hygromycin B selection. Results and discussion: Out of the total 617 co-cultivated calli, 21 (3.4%) regenerated shoots exhibited three distinct phenotypes: albino, chimeric, and pale green; in comparison to the wild-type non-transformed regenerated shoots. Total chlorophyll content was drastically reduced in albino shoots and significantly decreased in chimeric shoots. Out of the six Cas9 gene PCR-confirmed regenerated shoots, two exhibited the albino phenotype due to insertions/deletions (InDels) and substitution-based mutations in and around the AcPDS target sites. Deep amplicon sequencing revealed a significantly variable InDel frequency between two sgRNAs, ranging from 1.2% to 63.4%, along with a 53.4% substitution frequency. The mutation of the AcPDS gene generated a visually detectable albino phenotype, thus confirming the successful editing of the AcPDS gene. This is the first time a CRISPR/Cas9-mediated genome editing protocol has been successfully established in onion, with the AcPDS gene serving as an example. This study will provide the necessary momentum for researchers to further basic and applied research on onions.

Identification, cloning and characterization of AcMSH1 from Onion (Allium cepa L.).

BACKGROUND: MSH1 (MutS homolog1) is a nuclear-encoded protein that plays a crucial role in maintaining low mutation rates and stability of the organellar genome. While plastid MSH1 maintains nuclear epigenome plasticity and affects plant development patterns, mitochondrial MSH1 suppresses illegitimate recombination within the mitochondrial genome, affects mitochondrial genome substoichiometric shifting activity and induces cytoplasmic male sterility (CMS) in crops. However, a detailed functional investigation of onion MSH1 has yet to be achieved. MATERIALS AND RESULTS: The homology analysis of onion genome database identified a single copy of the AcMSH1 gene in the onion cv. Bhima Super. In silico analysis of AcMSH1 protein sequence revealed the presence of 6 conserved functional domains including a unique MSH1-specific GIY-YIG endonuclease domain at the C-terminal end. At N-terminal end, it has signal peptide sequences targeting chloroplast and mitochondria. The concentration of AcMSH1 was found to be highest in isolated mitochondria, followed by chloroplasts, and negligible in the cytoplasmic fraction; which proved its localization to the mitochondria and chloroplasts. Quantitative expression analysis revealed that AcMSH1 protein levels were highest in leaves, followed by flower buds, root tips, flowers, and umbels, with the lowest amount found in callus tissue. CONCLUSION: Onion genome has single copy of MSH1, with characteristic GIY-YIG endonuclease domain. AcMSH1 targeted towards both chloroplasts and mitochondria. The identification and characterisation of AcMSH1 may provide valuable insights into the development of CMS lines in onion.