Identification of Novel Loci Precisely Modulating Pre-Harvest Sprouting Resistance and Red Color Components of the Seed Coat in T. aestivum L.

The association between pre-harvest sprouting (PHS) and seed coat color has long been recognized. Red-grained wheats generally exhibit greater PHS resistance compared to white-grained wheat, although variability in PHS resistance exists within red-grained varieties. Here, we conducted a genome-wide association study on a panel consisting of red-grained wheat varieties, aimed at uncovering genes that modulate PHS resistance and red color components of seed coat using digital image processing. Twelve loci associated with PHS traits were identified, nine of which were described for the first time. Genetic loci marked by SNPs AX-95172164 (chromosome 1B) and AX-158544327 (chromosome 7D) explained approximately 25% of germination index variance, highlighting their value for breeding PHS-resistant varieties. The most promising candidate gene for PHS resistance was TraesCS6B02G147900, encoding a protein involved in aleurone layer morphogenesis. Twenty-six SNPs were significantly associated with grain color, independently of the known Tamyb10 gene. Most of them were related to multiple color characteristics. Prioritization of genes within the revealed loci identified TraesCS1D03G0758600 and TraesCS7B03G1296800, involved in the regulation of pigment biosynthesis and in controlling pigment accumulation. In conclusion, our study identifies new loci associated with grain color and germination index, providing insights into the genetic mechanisms underlying these traits.

Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells.

The auxin-inducible degron (AID) system is a promising tool for dynamic protein degradation. In mammalian cells, this approach has become indispensable to study fundamental molecular functions, such as replication, chromatin dynamics, or transcription, which are otherwise difficult to dissect. We present evaluation of the two prominent AID systems based on OsTIR1 and AtAFB2 auxin receptor F-box proteins (AFBs). We analyzed degradation dynamics of cohesin/condensin complex subunits in mouse embryonic stem cells (Rad21, Smc2, Ncaph, and Ncaph2) and human haploid HAP1 line (RAD21, SMC2). Double antibiotic selection helped achieve high homozygous AID tagging of an endogenous gene for all genes using CRISPR/Cas9. We found that the main challenge for successful protein degradation is obtaining cell clones with high and stable AFB expression levels due to the mosaic expression of AFBs. AFB expression from a transgene tends to decline with passages in the absence of constant antibiotic selection, preventing epigenetic silencing of a transgene, even at the AAVS1 safe-harbor locus. Comparing two AFBs, we found that the OsTIR1 system showed weak dynamics of protein degradation. At the same time, the AtAFB2 approach was very efficient even in random integration of AFB-expressed transgenes. Other factors such as degradation dynamics and low basal depletion were also in favor of the AtAFB2 system.