Transcriptomic response for revealing the molecular mechanism of oat flowering under different photoperiods.

Proper flowering is essential for the reproduction of all kinds of plants. Oat is an important cereal and forage crop; however, its cultivation is limited because it is a long-day plant. The molecular mechanism by which oats respond to different photoperiods is still unclear. In this study, oat plants were treated under long-day and short-day photoperiods for 10 days, 15 days, 20 days, 25 days, 30 days, 40 days and 50 days, respectively. Under the long-day treatment, oats entered the reproductive stage, while oats remained vegetative under the short-day treatment. Forty-two samples were subjected to RNA-Seq to compare the gene expression patterns of oat under long- and short-day photoperiods. A total of 634-5,974 differentially expressed genes (DEGs) were identified for each time point, while the floral organ primordium differentiation stage showed the largest number of DEGs, and the spikelet differentiation stage showed the smallest number. Gene Ontology (GO) analysis showed that the plant hormone signaling transduction and hormone metabolism processes significantly changed in the photoperiod regulation of flowering time in oat. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Mapman analysis revealed that the DEGs were mainly concentrated in the circadian rhythm, protein antenna pathways and sucrose metabolism process. Additionally, transcription factors (TFs) involved in various flowering pathways were explored. Combining all this information, we established a molecular model of oat flowering induced by a long-day photoperiod. Taken together, the long-day photoperiod has a large effect at both the morphological and transcriptomic levels, and these responses ultimately promote flowering in oat. Our findings expand the understanding of oat as a long-day plant, and the explored genes could be used in molecular breeding to help break its cultivation limitations in the future.

Transcriptome analysis of drought-responsive and drought-tolerant mechanisms in maize leaves under drought stress.

Maize is a major crop essential for food and feed, but its production is threatened by various biotic and abiotic stresses. Drought is one of the most common abiotic stresses, causing severe crop yield reduction. Although several studies have been devoted to selecting drought-tolerant maize lines and detecting the drought-responsive mechanism of maize, the transcriptomic differences between drought-tolerant and drought-susceptible maize lines are still largely unknown. In our study, RNA-seq was performed on leaves of the drought-tolerant line W9706 and the drought-susceptible line B73 after drought treatment. We identified 3147 differentially expressed genes (DEGs) between these two lines. The upregulated DEGs in W9706 were enriched in specific processes, including ABA signaling, wax biosynthesis, CHO metabolism, signal transduction and brassinosteroid biosynthesis-related processes, while the downregulated DEGs were enriched in specific processes, such as stomatal movement. Altogether, transcriptomic analysis suggests that the different drought resistances were correlated with the differential expression of genes, while the drought tolerance of W9706 is due to the more rapid response to stimulus, higher water retention capacity and stable cellular environment under water deficit conditions.

Mutation in Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase Gene ZmCRD1 Causes Chlorophyll-Deficiency in Maize.

Chlorophyll molecules are non-covalently associated with chlorophyll-binding proteins to harvest light and perform charge separation vital for energy conservation during photosynthetic electron transfer in photosynthesis for photosynthetic organisms. The present study characterized a pale-green leaf (pgl) maize mutant controlled by a single recessive gene causing chlorophyll reduction throughout the whole life cycle. Through positional mapping and complementation allelic test, Zm00001d008230 (ZmCRD1) with two missense mutations (p.A44T and p.T326M) was identified as the causal gene encoding magnesium-protoporphyrin IX monomethyl ester cyclase (MgPEC). Phylogenetic analysis of ZmCRD1 within and among species revealed that the p.T326M mutation was more likely to be causal. Subcellular localization showed that ZmCRD1 was targeted to chloroplasts. The pgl mutant showed a malformed chloroplast morphology and reduced number of starch grains in bundle sheath cells. The ZmCRD1 gene was mainly expressed in WT and mutant leaves, but the expression was reduced in the mutant. Most of the genes involved in chlorophyll biosynthesis, chlorophyll degradation, chloroplast development and photosynthesis were down-regulated in pgl. The photosynthetic capacity was limited along with developmental retardation and production reduction in pgl. These results confirmed the crucial role of ZmCRD1 in chlorophyll biosynthesis, chloroplast development and photosynthesis in maize.

A new strategy for using historical imbalanced yield data to conduct genome-wide association studies and develop genomic prediction models for wheat breeding.

Using imbalanced historical yield data to predict performance and select new lines is an arduous breeding task. Genome-wide association studies (GWAS) and high throughput genotyping based on sequencing techniques can increase prediction accuracy. An association mapping panel of 227 Texas elite (TXE) wheat breeding lines was used for GWAS and a training population to develop prediction models for grain yield selection. An imbalanced set of yield data collected from 102 environments (year-by-location) over 10 years, through testing yield in 40-66 lines each year at 6-14 locations with 38-41 lines repeated in the test in any two consecutive years, was used. Based on correlations among data from different environments within two adjacent years and heritability estimated in each environment, yield data from 87 environments were selected and assigned to two correlation-based groups. The yield best linear unbiased estimation (BLUE) from each group, along with reaction to greenbug and Hessian fly in each line, was used for GWAS to reveal genomic regions associated with yield and insect resistance. A total of 74 genomic regions were associated with grain yield and two of them were commonly detected in both correlation-based groups. Greenbug resistance in TXE lines was mainly controlled by Gb3 on chromosome 7DL in addition to two novel regions on 3DL and 6DS, and Hessian fly resistance was conferred by the region on 1AS. Genomic prediction models developed in two correlation-based groups were validated using a set of 105 new advanced breeding lines and the model from correlation-based group G2 was more reliable for prediction. This research not only identified genomic regions associated with yield and insect resistance but also established the method of using historical imbalanced breeding data to develop a genomic prediction model for crop improvement. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01287-8.

E183K Mutation in Chalcone Synthase C2 Causes Protein Aggregation and Maize Colorless.

Flavonoids give plants their rich colors and play roles in a number of physiological processes. In this study, we identified a novel colorless maize mutant showing reduced pigmentation throughout the whole life cycle by EMS mutagenesis. E183K mutation in maize chalcone synthase C2 (ZmC2) was mapped using MutMap strategy as the causal for colorless, which was further validated by transformation in Arabidopsis. We evaluated transcriptomic and metabolic changes in maize first sheaths caused by the mutation. The downstream biosynthesis was blocked while very few genes changed their expression pattern. ZmC2-E183 site is highly conserved in chalcone synthase among Plantae kingdom and within species' different varieties. Through prokaryotic expression, transient expression in maize leaf protoplasts and stable expression in Arabidopsis, we observed that E183K and other mutations on E183 could cause almost complete protein aggregation of chalcone synthase. Our findings will benefit the characterization of flavonoid biosynthesis and contribute to the body of knowledge on protein aggregation in plants.

Transcriptomic analysis reveals somatic embryogenesis-associated signaling pathways and gene expression regulation in maize (Zea mays L.).

KEY MESSAGE: Transcriptome analysis of maize embryogenic callus and somatic embryos reveals associated genes reprogramming, hormone signaling pathways and transcriptional regulation involved in somatic embryogenesis in maize. Somatic embryos are widely utilized in propagation and genetic engineering of crop plants. In our laboratory, an elite maize inbred line Y423 that could generate intact somatic embryos was obtained and applied to genetic transformation. To enhance our understanding of regulatory mechanisms during maize somatic embryogenesis, we used RNA-based sequencing (RNA-seq) to characterize the transcriptome of immature embryo (IE), embryogenic callus (EC) and somatic embryo (SE) from maize inbred line Y423. The number of differentially expressed genes (DEGs) in three pairwise comparisons (IE-vs-EC, IE-vs-SE and EC-vs-SE) was 5767, 7084 and 1065, respectively. The expression patterns of DEGs were separated into eight major clusters. Somatic embryogenesis associated genes were mainly grouped into cluster A or B with an expression trend toward up-regulation during dedifferentiation. GO annotation and KEGG pathway analysis revealed that DEGs were implicated in plant hormone signal transduction, stress response and metabolic process. Among the differentially expressed transcription factors, the most frequently represented families were associated with the common stress response or related to cell differentiation, embryogenic patterning and embryonic maturation processes. Genes include hormone response/transduction and stress response, as well as several transcription factors were discussed in this study, which may be potential candidates for further analyses regarding their roles in somatic embryogenesis. Furthermore, the temporal expression patterns of candidate genes were analyzed to reveal their roles in somatic embryogenesis. This transcriptomic data provide insights into future functional studies, which will facilitate further dissections of the molecular mechanisms that control maize somatic embryogenesis.

Assessment of the Potential for Genomic Selection To Improve Husk Traits in Maize.

Husk has multiple functions such as protecting ears from diseases, infection, and dehydration during development. Additionally, husks comprised of fewer, shorter, thinner, and narrower layers allow faster moisture evaporation of kernels prior to harvest. Intensive studies have been conducted to identify appropriate husk architecture by understanding the genetic basis of related traits, including husk length, husk layer number, husk thickness, and husk width. However, marker-assisted selection is inefficient because the identified quantitative trait loci and associated genetic loci could only explain a small proportion of total phenotypic variation. Genomic selection (GS) has been used successfully on many species including maize on other traits. Thus, the potential of using GS for husk traits to directly identify superior inbred lines, without knowing the specific underlying genetic loci, is well worth exploring. In this study, we compared four GS models on a maize association population with 498 inbred lines belonging to four subpopulations, including 27 lines in stiff stalk, 67 lines in non-stiff stalk, 193 lines in tropical-subtropical, and 211 lines in mixture subpopulations. Genomic Best Linear Unbiased Prediction with principal components as cofactor, performed the best and was selected to examine the impact of interaction between sampling proportions and subpopulations. We found that predictions on inbred lines in a subpopulation were benefited from excluding individuals from other subpopulations for training if the training population within the subpopulation was large enough. Husk thickness exhibited the highest prediction accuracy among all husk traits. These results gave strategic insight to improve husk architecture.

Genome-Wide Association Study Dissects the Genetic Architecture of Maize Husk Tightness.

The husk is a leafy outer tissue that encloses a maize ear. Previously, we identified the optimum husk structure by measuring the husk length, husk layer number, husk thickness and husk width. Husk tightness (HTI) is a combined trait based on the above four husk measurements. Unveiling the genetic basis of HTI will aid in guiding the genetic improvement of maize for mechanical harvesting and for protecting the ear from pest damage and pathogen infection. Here, we used a maize associate population of 508 inbred lines with tropical, subtropical and temperate backgrounds to analyze the genetic architecture of HTI. Evaluating the phenotypic diversity in three different environments showed that HTI exhibited broad natural variations and a moderate heritability level of 0.41. A diversity analysis indicated that the inbred lines having a temperate background were more loosely related than those having a tropical or subtropical background. HTI showed significant negative correlations with husk thickness and width, which indicates that thicker and wider husks wrapped the ear tighter than thinner and slimmer husks. Combining husk traits with ∼1.25 million single nucleotide polymorphisms in a genome-wide association study revealed 27 variants that were significantly associated with HTI above the threshold of P < 7.26 × 10-6. We found 27 candidate genes for HTI that may participate in (1) husk senescence involving lipid peroxidation (GRMZM2G017616) and programmed cell death (GRMZM2G168898 and GRMZM2G035045); (2) husk morphogenesis involving cell division (GRMZM5G869246) and cell wall architecture (GRMZM2G319798); and (3) cell signal transduction involving protein phosphorylation (GRMZM2G149277 and GRMZM2G004207) and the ABSISIC ACID INSENSITIVE3/VIVIPAROUS1 transcription factor (GRMZM2G088427). These results provide useful information for understanding the genetic basis of husk development. Further studies of identified candidate genes will help elucidate the molecular pathways that regulate HTI in maize.

Denser Markers and Advanced Statistical Method Identified More Genetic Loci Associated with Husk Traits in Maize.

The husk-the leaf-like outer covering of maize ear-has multiple functions, including protecting the ear from diseases infection and dehydration. In previous studies, we genotyped an association panel of 508 inbred lines genotyped with a total of ~550,000 SNPs (Illumina 50 K SNP Chip and RNA-seq). Genome-Wide Association Studies (GWAS) were conducted on four husk traits: husk length (HL), husk layer number (HN), husk thickness (HT), and husk width (HW). Minimal associations were identified and none of them passed the P-value threshold after a Bonferroni multiple-test correction using a single locus test in framework of mixed linear model. In this study, we doubled the number of SNPs (~1,250,000 in total) by adding GBS and 600 K SNP Chip. GWAS, performed with the recently developed multiple loci model (BLINK), revealed six genetic loci associated with HN and HT above the Bonferroni multiple-test threshold. Five candidate genes were identified based on the linkage disequilibrium with these loci, including GRMZM2G381691 and GRMZM2G012416. These two genes were up-regulation and down-regulation in all husk related tissues, respectively. GRMZM2G381691 associated with HT encoded a CCT domain protein, which expressed higher in tropical than temperate maize. GRMZM2G012416 associated with HN encoded an Armadillo (ARM) repeat protein, which regulated GA signal pathway. These associated SNPs and candidate genes paved a path to understand the genetic architecture of husk in maize.

Evaluation of the Potential for Genomic Selection to Improve Spring Wheat Resistance to Fusarium Head Blight in the Pacific Northwest.

Fusarium Head Blight (FHB) has emerged in spring wheat production in Pacific Northwest during the last decade due to factors including climate changes, crop rotations, and tillage practices. A breeding population with 170 spring wheat lines was established and screened over a 2-year period in multiple locations for FHB incidence (INC), severity (SEV), and deposition of the mycotoxin, deoxynivalenol (DON). A genome-wide association study suggested that the detectable number of genetic loci and effects are limited for marker-assisted selection. In conjunction with the success of breeding on FHB resistance in other programs, genomic selection (GS) was suggested as a better option. To evaluate the prediction accuracy of GS in the current breeding population, we conducted a variety of validations by varying proportions of testing populations and cohorts based on both FHB resistance and market class, including soft white spring (SWS), hard white spring (HWS), and hard red spring (HRS). We found that INC had higher heritability, higher correlation across years and locations, and higher prediction accuracy than SEV and DON. Prediction accuracy varied among the scenarios that restricted the testing population to a certain cohort. For a small set of newly developed or introduced lines (<17), prediction accuracy will be about 60% if the lines have similar genetic relationships as those among the current 170-line training population. However, we expect a lower prediction accuracy if new lines are selected for a specific characteristic, such as FHB resistance or market class. With the exception of DON in the SWS lines, the current training population is capable of making reasonably accurate predictions for FHB-resistant lines in most of the major market classes. For SWS, adding more lines or further phenotyping is required to improve prediction accuracy. These results demonstrate the potential and challenges of GS, especially for developing FHB-resistant varieties in the SWS market class.

Genome-Wide Association Study Reveals Novel Genes Associated with Culm Cellulose Content in Bread Wheat (Triticum aestivum, L.).

Plant cell wall formation is a complex, coordinated and developmentally regulated process. Cellulose is the most dominant constituent of plant cell walls. Because of its paracrystalline structure, cellulose is the main determinant of mechanical strength of plant tissues. As the most abundant polysaccharide on earth, it is also the focus of cellulosic biofuel industry. To reduce culm lodging in wheat and for improved ethanol production, delineation of the variation for stem cellulose content could prove useful. We present results on the analysis of the stem cellulose content of 288 diverse wheat accessions and its genome-wide association study (GWAS). Cellulose concentration ranged from 35 to 52% (w/w). Cellulose content was normally distributed in the accessions around a mean and median of 45% (w/w). Genome-wide marker-trait association study using 21,073 SNPs helped identify nine SNPs that were associated (p < 1E-05) with cellulose content. Four strongly associated (p < 8.17E-05) SNP markers were linked to wheat unigenes, which included ß-tubulin, Auxin-induced protein 5NG4, and a putative transmembrane protein of unknown function. These genes may be directly or indirectly involved in the formation of cellulose in wheat culms. GWAS results from this study have the potential for genetic manipulation of cellulose content in bread wheat and other small grain cereals to enhance culm strength and improve biofuel production.

Histological and transcript analyses of intact somatic embryos in an elite maize (Zea mays L.) inbred line Y423.

Intact somatic embryos were obtained from an elite maize inbred line Y423, bred in our laboratory. Using 13-day immature embryos after self-pollination as explants, and after 4-5 times subculture, a large number of somatic embryos were detected on the surface of the embryonic calli on the medium. The intact somatic embryos were transferred into the differential medium, where the plantlets regenerated with shoots and roots forming simultaneously. Histological analysis and scanning electron micrographs confirmed the different developmental stages of somatic embryogenesis, including globular-shaped embryo, pear-shaped embryo, scutiform embryo, and mature embryo. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used for comparative transcript profiling between embryogenic and non-embryogenic calli of a new elite maize inbred line Y423 during somatic embryogenesis. Differentially expressed genes were cloned and sequenced. Gene Ontology analysis of 117 candidate genes indicated their involvement in cellular component, biological process and molecular function. Nine of the candidate genes were selected. The changes in their expression levels during embryo induction and regeneration were analyzed in detail using quantitative real-time PCR. Two full-length cDNA sequences, encoding ZmSUF4 (suppressor of fir 4-like protein) and ZmDRP3A (dynamin-related protein), were cloned successfully from intact somatic embryos of the elite inbred maize line Y423. Here, a procedure for maize plant regeneration from somatic embryos is described. Additionally, the possible roles of some of these genes during the somatic embryogenesis has been discussed. This study is a systematic analysis of the cellular and molecular mechanism during the formation of intact somatic embryos in maize.