C. Stewart Gillmor

Summary: Defining the genetic basis of local adaptation is a key goal of evolutionary biology and crop improvement. Theory predicts that when selective pressures follow differences in the environment, a cline will be established. Clines can be exploited to uncover adaptive variation by association of alleles with the environment. However, monotonic phenotypic change over a cline is not necessarily mirrored in the behavior of genetic variants and population structure can further complicate analysis. To study genetic and phenotypic variation across the environment, we developed a multi‐parent advanced generation inter‐cross (MAGIC) population using eight Mexican native maize ( Zea mays L. ssp. mays ) varieties sourced from distinct agroecological zones. We evaluated the population in a common garden in Mexico and mapped tassel branching and flowering time, two traits that exhibit clinal variation. Variation in tassel branching was dominated by a single QTL with allele effects aligning to a negative elevational cline. By contrast, allele effects associated with 11 identified flowering time QTL were not consistently correlated with any one source environmental factor. Our observations support the prediction that genotype‐environment association will be strongest under simple genetic architecture, although, even then, analysis in native populations may be confounded by population structure.

Defining the genetic basis of local adaptation is fundamental to evolutionary biology and crop improvement. Theory predicts that when selective pressures track differences in the environment, a cline will be established. Such clines might be exploited to uncover adaptive variation by association of alleles with environmental stressors. However, monotonic phenotypic change over a cline is not necessarily mirrored by adaptive genetic variants. Furthermore, population structure can complicate the interpretation of genotype-environment association. To test the assumptions of genotype-environment association in a crop species, we developed a multi-parent advanced generation inter-cross (MAGIC) population using eight Mexican native maize varieties sourced from distinct agroecological zones. We mapped two clinal traits (tassel branching and flowering time) differing in genetic architecture. Variation in tassel branch number was dominated by a single QTL with allele effects that aligned well with a negative elevational cline. In contrast, we mapped 11 flowering time QTL with allele effects that were not consistently correlated with any one source environmental factor and distinct loci donated by highland and lowland early maturing varieties. Our observations support the theoretical result that genotype-environment association will be strongest under simple genetic architecture, although identification of adaptive alleles may still be confounded by population structure. Plain Language: Nine thousand years of careful selection and cultivation by indigenous farmers has generated a rich diversity of native Mexican maize (corn) varieties, grown from sea level to high mountains, and from jungle to semidesert. By crossing native varieties adapted to different locations, we can uncover important genetic variants conferring tolerance to environmental stressors.

Key message: Loss of SPL10 and SPL11 increases penetrance of abnormal phenotypes in dcl1 embryos. The first division of the Arabidopsis zygote is asymmetric, resulting in an apical cell lineage that generates most of the embryo proper, and a basal cell lineage that produces the root meristem and the extraembryonic suspensor. Loss of function mutations in the microRNA processing enzyme genes DICER-LIKE 1 (DCL1) and SERRATE (SE) show cell division defects in the embryo proper, hypophyseal cell, and suspensor. Previous transcriptome analyses showed that the microRNA156-targeted transcription factor genes SQUAMOSA PROMOTER BINDING PROTEIN-LIKE2 (SPL2) , SPL3 and SPL11 were upregulated in both globular stage dcl1 and se embryos, while SPL10 was upregulated in dcl1 . It was previously proposed that upregulation of SPL10 and SPL11 could explain some abnormal phenotypes in dcl1 embryos. In this work, we used T-DNA and CRISPR-Cas9-induced loss of function alleles to further explore the function of SPL2 , SPL3 , SPL10 and SPL11 in early embryogenesis and their contribution to the dcl1 phenotype. On their own, spl2 , spl3 , spl10 , and spl11 single mutants and an spl10 spl11 double mutant showed no abnormal cell divisions in early embryogenesis. In the dcl1/+ background, loss of function of SPL2 or SPL3 did not change the proportion of cell division defects in hypophyseal cells or suspensors observed in dcl1/+ . Loss of spl10 or spl11 in dcl1/+ resulted in a slight decrease or increase (respectively) in the penetrance of abnormal suspensor divisions in heart stage embryos, while the spl10 spl11 double mutant caused a small increase in the penetrance of abnormal hypophyseal divisions in dcl1 embryos. The differences between our results and previous studies are likely due to genetic redundancy of miR156-targeted SPL genes, variable environmental conditions or the effect of genetic background on the penetrance of the dcl1 phenotype. In the future, analysis of higher order mutations in SPL and MIR156 genes will help to better understand the role of these important developmental regulators in early embryo development.

After fertilization in animals, maternal mRNAs and proteins regulate development until the onset of zygotic transcription. In plants, the extent of maternal regulation of early embryo development has been less clear: two hybrid combinations of rice zygotes have a strong maternal transcript bias, zygotes of a third rice hybrid produced by gamete fusion show a small percentage of maternally biased genes, while Arabidopsis Col/Cvi and Col/Ler hybrid embryos display symmetric and asymmetric parental genome activation, respectively. Here, we explore parent-of-origin transcriptome behavior in the Arabidopsis Col/Tsu hybrid, which was previously shown to display maternal effects for embryo defective mutants indistinguishable from those of the reference ecotype, Col. Analysis of Col/Tsu transcriptomes revealed a reciprocal maternal bias in thousands of genes in zygotes and octant stage embryos. Several lines of evidence suggest that this transient maternal bias is due to preferential transcription of maternal alleles in the zygote, rather than inheritance of transcripts from the egg. Our results extend previous observations that parent-of-origin contributions to early embryogenesis differ between hybrids of Arabidopsis, show that the maternal genome plays a predominant role in early embryos of Col/Tsu, and point to a maternal transcriptome bias in early embryos of the Arabidopsis reference ecotype Columbia.

Background: Plant long non-coding RNAs (lncRNAs) have important regulatory roles in responses to various biotic and abiotic stresses, including light quality. However, no lncRNAs have been specifically linked to the Shade Avoidance Response (SAS). Results: To better understand the involvement of lncRNAs in shade avoidance, we examined RNA-seq libraries for lncRNAs with the potential to function in the neighbor proximity phenomenon in Arabidopsis thaliana (A. thaliana). Using transcriptomes generated from seedlings exposed to high and low red/far-red (R/FR) light conditions, we identified 13 lncRNA genes differentially expressed in cotyledons and 138 in hypocotyls. To infer possible functions for these lncRNAs, we used a ‘guilt-by-association’ approach to identify genes co-expressed with lncRNAs in a weighted gene co-expression network. Of 34 co-expression modules, 10 showed biological functions related to differential growth. We identified three potential lncRNAs co-regulated with genes related to SAS. T-DNA insertions in two of these lncRNAs were correlated with morphological differences in seedling responses to increased FR light, supporting our strategy for computational identification of lncRNAs involved in SAS. Conclusions: Using a computational approach, we identified multiple lncRNAs in Arabidopsis involved in SAS. T-DNA insertions caused altered phenotypes under low R/FR light, suggesting functional roles in shade avoidance. Further experiments are needed to determine the specific mechanisms of these lncRNAs in SAS.

After fertilization, zygotic genome activation results in a transcriptionally competent embryo. Hybrid transcriptome experiments in Arabidopsis have concluded that the maternal and paternal genomes make equal contributions to zygotes and embryos, yet embryo defective (emb) mutants in the Columbia (Col) ecotype display early maternal effects. Here, we show that hybridization of Col with Landsberg erecta (Ler) or Cape Verde Islands (Cvi) ecotypes decreases the maternal effects of emb mutants. Reanalysis of Col/Ler and Col/Cvi transcriptomes confirmed equal parental contributions in Col/Cvi early embryos. By contrast, thousands of genes in Col/Ler zygotes and one-cell embryos were biallelic in one cross and monoallelic in the reciprocal cross, with analysis of intron reads pointing to active transcription as responsible for this parent-of-origin bias. Our analysis shows that, contrary to previous conclusions, the maternal and paternal genomes in Col/Ler zygotes are activated in an asymmetric manner. The decrease in maternal effects in hybrid embryos compared with those in isogenic Col along with differences in genome activation between Col/Cvi and Col/Ler suggest that neither of these hybrids accurately reflects the general trends of parent-of-origin regulation in Arabidopsis embryogenesis.

Plant PHO1 proteins play a central role in the translocation and sensing of inorganic phosphate. The maize (Zea maysssp.mays) genome encodes two co‐orthologs of theArabidopsis PHO1gene, designatedZmPho1;2aandZmPho1;2b. Here, we report the characterization of the transposon footprint alleleZmpho1;2a′‐m1.1, which we refer to hereafter aspho1;2a. Thepho1;2aallele is a stable derivative formed by excision of anActivatortransposable element from theZmPho1;2agene. Thepho1;2aallele contains an 8‐bp insertion at the point of transposon excision that disrupts the reading frame and is predicted to generate a premature translational stop. We show that thepho1;2aallele is linked to a dosage‐dependent reduction inPho1;2atranscript accumulation and a mild reduction in seedling growth. Characterization of shoot and root transcriptomes under full nutrient, low nitrogen, low phosphorus, and combined low nitrogen and low phosphorus conditions identified 1100 differentially expressed genes between wild‐type plants and plants carrying thepho1;2amutation. Of these 1100 genes, 966 were upregulated in plants carryingpho1;2a, indicating the wild‐type PHO1;2a to predominantly impact negative gene regulation. Gene set enrichment analysis of thepho1;2a‐misregulated genes revealed associations with phytohormone signaling and the phosphate starvation response. In roots, differential expression was broadly consistent across all nutrient conditions. In leaves, differential expression was largely specific to low phosphorus and combined low nitrogen and low phosphorus conditions. Of 276 genes upregulated in the leaves ofpho1;2amutants in the low phosphorus condition, 153 were themselves induced in wild‐type plants with respect to the full nutrient condition. Our observations suggest thatPho1;2afunctions in the fine‐tuning of the transcriptional response to phosphate starvation through maintenance and/or sensing of plant phosphate status.

PHO1 proteins play a central role in plant inorganic phosphorus translocation and sensing. The maize ( Zea mays ssp. mays ) genome encodes two co-orthologs of the Arabidopsis PHO1 gene, designated ZmPho1;2a and ZmPho1;2b . Here, we report the characterization of the transposon-footprint allele Zmpho1;2a’-m1 . 1 , which we refer to hereafter as pho1;2a . The pho1;2a allele is a stable derivative formed by excision of an Activator element from the ZmPho1;2a gene. The pho1;2a allele contains an 8 bp insertion at the point of excision that disrupts the reading frame and is predicted to generate a premature translational stop. We show that the pho1;2a allele is linked to a dosage-dependent reduction in transcript accumulation and a mild reduction in seedling growth that is enhanced under nutrient deficient conditions. Characterization of the shoot and root transcriptomes of seedlings segregating the pho1;2a mutation under different nutrient conditions revealed pho1;2a to have a dominant effect on patterns of transcript accumulation. Gene set enrichment analysis of the transcripts mis-regulated in pho1;2a mutants suggests that Pho1;2a functions in the fine-tuning of the transcriptional phosphate starvation response. We discuss our results with reference to possible genetic redundancy among maize Pho1 genes and in the context of reports linking functional variation in Pho1;2a to agronomically important traits.