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Genetic and Molecular Determinants of Spikelet Survival in Cereal Crops

Periodic Reporting for period 3 - LUSH SPIKE (Genetic and Molecular Determinants of Spikelet Survival in Cereal Crops)

Reporting period: 2019-07-01 to 2020-12-31

Meeting the forecasted world demand for food remains a crucial challenge for plant scientists in this century. One promising avenue for improving grain yield of cereal crops, including wheat and barley, involves reducing spikelet mortality. Spikelets, the grain-bearing units of cereal spikes, usually form in excess and subsequently abort during development; increased spikelet survival is linked to increased numbers of grains per spike. Therefore, reducing spikelet mortality is an intriguing approach to improve grain yield.

In barley, the number of spikelets per spike at the awn primordium (AP) stage represents the maximum yield potential per spike. After the AP stage, significant spikelet mortality results in fewer grains per spike. Our previous results clearly indicated that spikelet survival in barley is highly genetically controlled (broad-sense heritability >0.80) and that the period from AP to tipping represents the most critical pre-anthesis phase related to spikelet reduction and grain yield per spike. However, the underlying genetic and molecular determinants of spikelet survival remain to be discovered. I therefore propose this ambitious research program with an emphasis on using available genetic resources.

Our specific aims during the LUSH SPIKE project are to:
(i) discover quantitative trait loci (QTL) for spikelet survival and grain number per spike and validate these QTL in bi-parental doubled-haploid mapping populations,
(ii) isolate and functionally characterize Mendelized QTL using a map-based approach,
(iii) reveal gene regulatory networks determining spikelet survival during the critical spike growth period from AP to heading, and
(iv) elucidate spatio-temporal patterns of metabolite and phytohormone distributions in spike and spikelet sections during the critical growth period, using mass spectrometric imaging.

The results we obtain will advance our understanding of how to improve yields of cereal crops.
Ad i+ii.) We refined our phenotypic analyses during the reproductive life cycle of barley plants following the so called WADDINGTON (W) scale (Waddington et al. 1983; Ann Bot). We therefore 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 following the W scale. We show that the spikelet abortion process can be divided into several phases regardless of growth conditions (Thirulogachandar, Koppolu et al.; in preparation).
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. All accessions have been sequenced with low genome coverage using GBS in the frame work of a different project, which had characterized the entire IPK barley collection (Milner et al. 2018; Nat Genet). The rationale of selecting an almost unstructured GWAS panel for QTL identification will allow having a shortcut to the gene(s) of interest without using the lengthy and more tedious map-based cloning strategy. Moreover, it allows assaying more alleles at the same time and may provide a much better overview of how many major/minor QTLs are present in the panel. DH populations may then be used for validating identified QTLs from GWAS.
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. For ~125 accessions we did not have enough grains and multiplied them in the GH instead; however, they will become available during the field season in 2019. Even though we only used 275 accessions for spikelet survival and in total ca. 15,000 SNP markers, we were able to detect first QTLs on several chromosomes (Kamal Kaur et al.; in preparation). Now, these QTL must be validated in upcoming field seasons 2019 and 2020; but using the whole panel of ~400 accessions and more markers (i.e. we are planning to sequence all ~400 accessions with 2-3x genome coverage (barley 5 Gbp) following a whole-genome shotgun, WGS, approach). We assume that by using WGS-sequencing we may be able to increase the number of markers by a factor of 10, which in turn may facilitate an improved GWAS analysis via haplotype blocks. Moreover, all ~400 accessions will undergo detailed phenotypic analyses under controlled growth conditions during the fall/winter/early spring periods of the years 2018-19 and 2019-20.

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.; in preparation), 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 at ~W4.5-5.5 (Thirulogachandar, Rutten et al.; in preparation). We therefore found transcript signatures for the onset of abortion/mitotic arrest in our available data set. Established GRNs are under continuous investigations (Koppolu et al.; in preparation).

Ad iv.) The distribution of metabolites and phytohormones in space and time during pre-anthesis spike development is largely unknown in cereal crops; however, our previous work was suggestive of hormonal gradients along the barley spike (Youssef et al. 2017; Nat Genet). To improve our understanding of hormonal and metabolite regulation of yield-establishing processes, such as spikelet decline, we proposed within the current 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.
To this end, we investigated immature barley spike sections (apical, central, basal) for various phytohormones, i.e. Auxin (IAA), Cytokinin (trans-zeatin, CK), Abscisic Acid (ABA), Jasmonic Acid (JA) and Salicylic Acid (SA) over several time course experiments. We monitored hormonal concentrations and distributions in spike sections during the critical period from the onset of mitotic arrest until the visible apical spikelet abortion. Since the onset of spikelet abortion starts in the most apical spike parts first, progressing basipetally over time; the apical spike sections are highly relevant in terms of hormonal concentrations. We found that IAA and CK concentrations declined over time from ~W4.0 to W7.0 while ABA was the only hormone, which increased during the critical period only in the apical spike part, suggesting that ABA is involved in the spikelet abortion process (Shanmugaraj et al.; in preparation). First MS-imaging results for amino acid distribution in immature spike sections are promising.