Ad i+ii.) We phenotypically analyzed in total 29 genotypes of spring barley (all of which are parents of our DH mapping populations) under controlled growth (i.e. greenhouse, GH) and field conditions for their growth curve patterns. We show that the spikelet abortion process can be divided into several phases regardless of growth conditions (Thirulogachandar et al. 2021). For QTL discovery, we selected a diverse GWAS panel of six-rowed barleys (~400 accessions) based on high genotypic diversity; all accessions were selected for photoperiod sensitivity alleles at the major photoperiod locus Ppd-H1. In 2018, we already had 275 out of the ~400 accessions in a field trial (double rows per accession) and investigated traits such as maximum yield potential, spikelet survival, grain number per spike and other agronomically relevant characters for initial GWAS analyses (Kamal et al. 2022). QTL were validated in field seasons 2018 to 2020 using the whole panel of ~400 accessions and more markers (i.e. ~22 Mio from 2-3x genome coverage; following a whole-genome shotgun, WGS, approach). Moreover, 358 accessions have undergone detailed phenotypic analyses under controlled growth conditions (GH) during the fall/winter/early spring periods of the years 2018-19 and 2019-20. For both environments, field and GH, we found highly significant QTLs almost to single-gene resolution. Functional gene validation work is underway for three QTL regions (Huang et al.; in preparation/ Kamal et al.; in preparation).
Ad iii.) From our previous works related to the generation of a barley spike transcript atlas using laser-aided micro dissections of meristems (Thiel, Koppolu et al. 2021), we discovered that we had already covered the critical period for the initiation of spikelet abortion. Visible spikelet abortion is rather the end point of an occurring mitotic arrest of the apical inflorescence meristem, which starts on the cellular level much earlier. We therefore found transcript signatures for the onset of abortion/mitotic arrest in our available data set. Established GRNs are still under continuous investigations (Huang et al.; in preparation).
Ad iv.) To improve our understanding of hormonal and metabolite regulation of yield-establishing processes, such as spikelet decline, we established within the LUSH SPIKE project a detailed histological and microscopic analysis using the latest mass spectrometric imaging techniques (Peukert et al. 2014; Plant Cell) to integrate metabolite and hormonal analyses with organ growth over the critical period of development. Using such MALDI-MS imaging, we found for the first time the spatio-temporal distribution of important metabolites, such as sugars, amino acids and the phytohormone melatonin in developing barley spikes. By combining metabolomic, transcriptomic, and genetic approaches we show that apical abortion is associated with sugar depletion, amino acid degradation, and ABA biosynthesis and signaling. Senescence and defense-responsive transcription factor families, viz., NACs, HD-ZIPs, bZIPs, and MYBs, are amongst the putative candidate genes responsible for apical abortion. CRISPR/Cas9-mediated knock-out of barley GRASSY TILLERS1 (HvGT1) encoding an HD-ZIP transcription factor delayed apical abortion, thereby increasing the final spikelet number. We thus propose that modifying apical spikelet abortion by exploiting the identified putative regulators may help increase yield potential in barley and other related cereals (Shanmugaraj et al.; in preparation).
