Haploid induction by nanobody-targeted ubiquitin-proteasome-based degradation of EYFP-tagged CENH3 in Arabidopsis thaliana.

The generation of haploid plants accelerates the crop breeding process. One of the haploidization strategies is based on the genetic manipulation of endogenous centromere-specific histone 3 (CENH3). To extend the haploidization toolbox, we tested whether targeted in vivo degradation of CENH3 protein can be harnessed to generate haploids in Arabidopsis thaliana. We show that a recombinant anti-GFP nanobody fused to either heterologous F-box (NSlmb) or SPOP/BTB ligase proteins can recognize maternally derived enhanced yellow fluorescent protein (EYFP)-tagged CENH3 in planta and make it accessible for the ubiquitin-proteasome pathway. Outcrossing of the genomic CENH3-EYFP-complemented cenh3.1 mother with plants expressing the GFP-nanobody-targeted E3 ubiquitin ligase resulted in a haploid frequency of up to 7.6% in pooled F1 seeds. EYFP-CENH3 degradation occurred independently in embryo and endosperm cells. In reciprocal crosses, no haploid induction occurred. We propose that the uniparental degradation of EYFP-fused genomic CENH3 during early embryogenesis leads to a decrease in its level at centromeres and subsequently weakens the centromeres. The male-derived wild type CENH3 containing centromere outcompetes the CENH3-EYFP depleted centromere. Consequently, maternal chromosomes undergo elimination, resulting in haploids.

Optimization of quantitative reverse transcription PCR method for analysis of weakly expressed genes in crops based on rapeseed.

Rapeseed (Brassica napus) is an allopolyploid hybrid (AACC genome) of turnip rape (B. rapa, genome: AA) and vegetable cabbage (B. oleraceae, genome: CC). Rapeseed oil is one of the main vegetable oils used worldwide for food and other technical purposes. Therefore, breeding companies worldwide are interested in developing rapeseed varieties with high yields and increased adaptation to harsh climatic conditions such as heat and prolonged drought. One approach to studying the mechanism of the epigenetically regulated stress response is to analyze the transcriptional changes it causes. In addition, comparing the expression of certain genes between stress- and non-stress-tolerant varieties will help guide breeding in the desired direction. Quantitative reverse transcription PCR (RT-qPCR) has been intensively used for gene expression analysis for several decades. However, the transfer of this method from model plants to crop species has several limitations due to the high accumulation of secondary metabolites, the higher water content in some tissues and therefore problems with their grinding and other factors. For allopolyploid rapeseed, the presence of two genomes, often with different levels of expression of homeologous genes, must also be considered. In this study, we describe the optimization of transcriptional RT-qPCR analysis of low-expression epigenetic genes in rapeseed, using Kinetochore Null2 (KNL2), a regulator of kinetochore complex assembly, as an example. We demonstrated that a combination of various factors, such as tissue homogenization and RNA extraction with TRIzol, synthesis of cDNA with gene-specific primers, and RT-qPCR in white plates, significantly increased the sensitivity of RT-qPCR for the detection of BnKNL2A and BnKNL2C gene expression.