Lines with Substituted Chimeric GFP-CENH3 Give Haploid and Aneuploid Progenies on Crossing with Other Lines.

Haploids and doubled haploids are invaluable for basic genetic studies and in crop improvement. A novel method of haploid induction through genetic engineering of the Centromere Histone Protein gene, , has been demonstrated in Arabidopsis. The present study was undertaken to develop haploid inducer (HI) lines of based on the principles elaborated in Arabidopsis. was found to carry three copies of which generated five different transcripts, of which three transcripts resulted from alternative splicing. Unlike where native gene was knocked out for constructing HI lines, we used RNAi approach to knockdown the native genes. Further, to rescue CENH3 silenced cells, a GFP-CENH3-tailswap construct having N terminal GFP fused to H3.3 tail sequences and synthetic CENH3 histone fold domain sequences was devised. A total 38 transgenic plants were regenerated following co-transformation with both silencing and rescue cassettes and transgenics carrying either or both the constructs were obtained. Transgenic status was confirmed through PCR, Southern and qRT-PCR analyses. Co-transformed lines were crossed to untransformed or a line expressing only GFP-tailswap. FACS and cytological analyses of progenies revealed partial or complete elimination of chromosomes thereby giving rise to aneuploids and haploid. This is the first report in a polyploid crop demonstrating that CENH3 engineering could be used to develop HI lines.