Identification and Analysis of lncRNA and circRNA Related to Wheat Grain Development.

The role of lncRNA and circRNA in wheat grain development is still unclear. The objectives of this study were to characterize the lncRNA and circRNA in the wheat grain development and to construct the interaction network among lncRNA, circRNA, and their target miRNA to propose a lncRNA-circRNA-miRNA module related to wheat grain development. Full transcriptome sequencing on two wheat varieties (Annong 0942 and Anke 2005) with significant differences in 1000-grain weight at 10 d (days after pollination), 20 d, and 30 d of grain development were conducted. We detected 650, 736, and 609 differentially expressed lncRNA genes, and 769, 1054, and 1062 differentially expressed circRNA genes in the grains of 10 days, 20 days and 30 days after pollination between Annong 0942 and Anke 2005, respectively. An analysis of the lncRNA-miRNA and circRNA-miRNA targeting networks reveals that circRNAs exhibit a more complex and extensive interaction network in the development of cereal grains and the formation of grain shape. Central to these interactions are tae-miR1177, tae-miR1128, and tae-miR1130b-3p. In contrast, lncRNA genes only form a singular network centered around tae-miR1133 and tae-miR5175-5p when comparing between varieties. Further analysis is conducted on the underlying genes of all target miRNAs, we identified TaNF-YB1 targeted by tae-miR1122a and TaTGW-7B targeted by miR1130a as two pivotal regulatory genes in the development of wheat grains. The quantitative real-time PCR (qRT-PCR) and dual-luciferase reporter assays confirmed the target regulatory relationships between miR1130a-TaTGW-7B and miR1122a-TaNF-YB1. We propose a network of circRNA and miRNA-mediated gene regulation in the development of wheat grains.

CRISPR/Cas9-mediated editing of Wx and BADH2 genes created glutinous and aromatic two-line hybrid rice.

Amylose content (AC) is one of the physicochemical indexes of rice quality, which is largely determined by the Waxy (Wx) gene. Fragrance in rice is favored because it adds good flavor and a faint scent. Loss of function of the BADH2 (FGR) gene promotes the biosynthesis of 2-acetyl-1-pyrroline (2AP), which is the main compound responsible for aroma in rice. Here, we used a CRISPR/Cas9 system to simultaneously knock out Wx and FGR genes in 1892S and M858, which are the parents of an indica two-line hybrid rice, Huiliangyou 858 (HLY858). Four T-DNA-free homozygous mutants (1892Swxfgr-1, 1892Swxfgr-2, M858wxfgr-1, and M858wxfgr-2) were obtained. The 1892Swxfgr and M858wxfgr were crossed to generate double mutant hybrid lines HLY858wxfgr-1 and HLY858wxfgr-2. Size-exclusion chromatography (SEC) data indicated that true AC of the wx mutant starches ranged from 0.22 to 1.63%, much lower than those of the wild types (12.93 to 13.76%). However, the gelatinization temperature (GT) of the wx mutants in backgrounds of 1892S, M858, and HLY858 were still high, and showed no significant differences with the wild type controls. The aroma compounds 2AP content in grains of HLY858wxfgr-1 and HLY858wxfgr-2 were 153.0 µg/kg and 151.0 µg/kg, respectively. In contrast, 2AP was not detected in grains of HLY858. There were no significant differences in major agronomic traits between the mutants and HLY858. This study provides guidelines for cultivation of ideal glutinous and aromatic hybrid rice by gene editing.

N6-Methyladenosine dynamic changes and differential methylation in wheat grain development.

MAIN CONCLUSION: More methylation changes occur in late interval than in early interval of wheat seed development with protein and the starch synthesis-related pathway enriched in the later stages. Wheat seed development is a critical process to determining wheat yield and quality, which is controlled by genetics, epigenetics and environments. The N6-methyladenosine (m6A) modification is a reversible and dynamic process and plays regulatory role in plant development and stress responses. To better understand the role of m6A in wheat grain development, we characterized the m6A modification at 10 day post-anthesis (DPA), 20 DPA and 30 DPA in wheat grain development. m6A-seq identified 30,615, 30,326, 27,676 high confidence m6A peaks from the 10DPA, 20DPA, and 30DPA, respectively, and enriched at 3'UTR. There were 29,964, 29,542 and 26,834 unique peaks identified in AN0942_10d, AN0942_20d and AN0942_30d. One hundred and forty-two genes were methylated by m6A throughout seed development, 940 genes methylated in early grain development (AN0942_20d vs AN0942_10d), 1542 genes in late grain development (AN0942_30d vs AN0942_20d), and 1190 genes between early and late development stage (AN0942_30d vs AN0942_10d). KEGG enrichment analysis found that protein-related pathways and the starch synthesis-related pathway were significantly enriched in the later stages of seed development. Our results provide novel knowledge on m6A dynamic changes and its roles in wheat grain development.

MiR396 regulatory network and its expression during grain development in wheat.

Wheat contains the largest number of miR396 family with 17 miR396 in Poaceae. MiR396 regulatory network underlying wheat grain development has not comprehensively been explored. Our results showed that precursor miR396 family in Poaceae exhibited not only conservativeness but also diversification especially in wheat. Five haplotypes were detected in Poaceae species, while 4 haplotypes in wheat with Hap-4 (miR396a) and Hap-5 (miR396n) unique to wheat. GO enrichment analysis of target genes showed that the first 20 enrichment functions of miR396a and miR396n are completely different from each other, and also completely different from miR396(b-g), miR396(h-m), and miR396(o-q). Functional annotation on the 18 target genes shared by miR396(b-g), miR396(h-m), and miR396(o-q) found that 11 of the 18 target genes are growth-regulating factor (GRF) genes. Our results indicated that, during the grain filling stage of wheat, miR396 is involved in the development of grains by regulating the expression of GRF genes (GRF1, GRF6, and GRF9). Although the enrichment function of miR396(b-g), miR396(h-m), and miR396(o-q) is the same, the gene functional networks they formed differ greatly. Our results indicated that polyploidization enriches not only the diversity of miR396 family and its target genes but also gene functional networks in wheat. These results laid foundation for further elucidating function of miR396 gene family underlying wheat grain development.

Physiological and transcriptional response to heat stress in heat-resistant and heat-sensitive maize (Zea mays L.) inbred lines at seedling stage.

To understand the molecular and physiological mechanism underlying the heat stress in maize, transcriptional and physiological response to heat stress in the heat-resistant Huangzaosi (HZS) and heat-sensitive Lv-9-Kuan (L9K) inbred lines at seedling stage were analyzed and compared at seedling stage. Our results indicated that MDA content of the two inbred lines increased significantly under heat stress; the values of MDA in L9K was significantly higher than that in HZS. The level of SOD, CAT, and POD enzyme activities in HZS was higher than those in L9K for both the heat-treated group and controls. The values of Fv/Fm, qP, and ФPSII reduced by heat stress in L9K were higher than the respective values in HZS. RNA-seq data showed that heat stress induced more heat stress-related genes in HZS (257 heat stress-related genes) than in L9K (224 heat stress-related genes). GO and KEGG enrichment analyses indicated that HZS and L9K changed their physiological and biochemical mechanisms in response to heat stress through different molecular mechanisms. Weighted Gene Co-expression Network Analysis showed that HZS might obtain stronger heat resistance than L9K through a unique transcriptional regulatory network. Our findings provide insights into the molecular networks that mediate the tolerance of maize heat stress and also help us to mine key heat stress-related genes.

The trihelix transcription factor OsGTγ-2 is involved adaption to salt stress in rice.

KEY MESSAGE: OsGTγ-2, a trihelix transcription factor, is a positive regulator of rice responses to salt stress by regulating the expression of ion transporters. Salinity stress seriously restricts rice growth and yield. Trihelix transcription factors (GT factors) specifically bind to GT elements and play a diverse role in plant morphological development and responses to abiotic stresses. In our previous study, we found that the GT-1 element (GAAAAA) is a key element in the salinity-induced OsRAV2 promoter. Here, we identified a rice OsGTγ family member, OsGTγ-2, which directly interacted with the GT-1 element in the OsRAV2 promoter. OsGTγ-2 specifically targeted the nucleus, was mainly expressed in roots, sheathes, stems and seeds, and was induced by salinity, osmotic and oxidative stresses and abscisic acid (ABA). The seed germination rate, seedling growth and survival rate under salinity stress was improved in OsGTγ-2 overexpressing lines (PZmUbi::OsGTγ-2). In contrast, CRISPR/Cas9-mediated OsGTγ-2 knockout lines (osgtγ-2) showed salt-hypersensitive phenotypes. In response to salt stress, different Na+ and K+ acclamation patterns were observed in PZmUbi::OsGTγ-2 lines and osgtγ-2 plants were observed. The molecular mechanism of OsGTγ-2 in rice salt adaptation was also investigated. Several major genes responsible for ion transporting, such as the OsHKT2; 1, OsHKT1; 3 and OsNHX1 were transcriptionally regulated by OsGTγ-2. A subsequent yeast one-hybrid assay and EMSA indicated that OsGTγ-2 directly interacted with the promoters of OsHKT2; 1, OsNHX1 and OsHKT1; 3. Taken together, these results suggest that OsGTγ-2 is an important positive regulator involved in rice responses to salt stress and suggest a potential role for OsGTγ-2 in regulating salinity adaptation in rice.

MicroRNA-mediated responses to colchicine treatment in barley.

MAIN CONCLUSION: In Hordeum vulgare, nine differentially expressed novel miRNAs were induced by colchicine. Five novel miRNA in colchicine solution showed the opposite expression patterns as those in water. Colchicine is a commonly used agent for plant chromosome set doubling. MicroRNA-mediated responses to colchicine treatment in plants have not been characterized. Here, we characterized new microRNAs induced by colchicine treatment in Hordeum vulgare using high-throughput sequencing. Our results showed that 39 differentially expressed miRNAs were affected by water treatment, including 34 novel miRNAs and 5 known miRNAs; 42 miRNAs, including 37 novel miRNAs and 5 known miRNAs, were synergistically affected by colchicine and water, and 9 differentially expressed novel miRNAs were induced by colchicine. The novel_mir69, novel_mir57, novel_mir75, novel_mir38, and novel_mir56 in colchicine treatment showed the opposite expression patterns as those in water. By analyzing these 9 differentially expressed novel miRNAs and their targets, we found that novel_mir69, novel_mir56 and novel_mir25 co-target the genes involving the DNA repair pathway. Based on our results, microRNA-target regulation network under colchicine treatment was proposed, which involves actin, cell cycle regulation, cell wall synthesis, and the regulation of oxidative stress. Overall, the results demonstrated the critical role of microRNAs mediated responses to colchicine treatment in plants.

Nucleotide diversity patterns at the DREB1 transcriptional factor gene in the genome donor species of wheat (Triticum aestivum L).

Bread wheat (AABBDD) originated from the diploid progenitor Triticum urartu (AA), a relative of Aegilops speltoides (BB), and Ae. tauschii (DD). The DREB1 transcriptional factor plays key regulatory role in low-temperature tolerance. The modern breeding strategies resulted in serious decrease of the agricultural biodiversity, which led to a loss of elite genes underlying abiotic stress tolerance in crops. However, knowledge of this gene's natural diversity is largely unknown in the genome donor species of wheat. We characterized the dehydration response element binding protein 1 (DREB1) gene-diversity pattern in Ae. speltoides, Ae. tauschii, T. monococcum and T. urartu. The highest nucleotide diversity value was detected in Ae. speltoides, followed by Ae. tauschii and T. monococcum. The lowest nucleotide diversity value was observed in T. urartu. Nucleotide diversity and haplotype data might suggest no reduction of nucleotide diversity during T. monococcum domestication. Alignment of the 68 DREB1 sequences found a large-size (70 bp) insertion/deletion in the accession PI486264 of Ae. speltoides, which was different from the copy of sequences from other accessions of Ae. speltoides, suggesting a likely existence of two different ancestral Ae. speltoides forms. Implication of sequences variation of Ae. speltoides on origination of B genome in wheat was discussed.

Origin and Evolution of Allopolyploid Wheatgrass Elymus fibrosus (Schrenk) Tzvelev (Poaceae: Triticeae) Reveals the Effect of Its Origination on Genetic Diversity.

Origin and evolution of tetraploid Elymus fibrosus (Schrenk) Tzvelev were characterized using low-copy nuclear gene Rpb2 (the second largest subunit of RNA polymerase II), and chloroplast region trnL-trnF (spacer between the tRNA Leu (UAA) gene and the tRNA-Phe (GAA) gene). Ten accessions of E. fibrosus along with 19 Elymus species with StH genomic constitution and diploid species in the tribe Triticeae were analyzed. Chloroplast trnL-trnF sequence data suggested that Pseudoroegneria (St genome) was the maternal donor of E. fibrosus. Rpb2 data confirmed the presence of StH genomes in E. fibrosus, and suggested that St and H genomes in E. fibrosus each is more likely originated from single gene pool. Single origin of E. fibrosus might be one of the reasons causing genetic diversity in E. fibrosus lower than those in E. caninus and E. trachycaulus, which have similar ecological preferences and breeding systems with E. fibrosus, and each was originated from multiple sources. Convergent evolution of St and H copy Rpb2 sequences in some accessions of E. fibrosus might have occurred during the evolutionary history of this allotetraploid.

Origin and Reticulate Evolutionary Process of Wheatgrass Elymus trachycaulus (Triticeae: Poaceae).

To study origin and evolutionary dynamics of tetraploid Elymus trachycaulus that has been cytologically defined as containing StH genomes, thirteen accessions of E. trachycaulus were analyzed using two low-copy nuclear gene Pepc (phosphoenolpyruvate carboxylase) and Rpb2 (the second largest subunit of RNA polymerase II), and one chloroplast region trnL-trnF (spacer between the tRNA Leu (UAA) gene and the tRNA-Phe (GAA) gene). Our chloroplast data indicated that Pseudoroegneria (St genome) was the maternal donor of E. trachycaulus. Rpb2 data indicated that the St genome in E. trachycaulus was originated from either P. strigosa, P. stipifolia, P. spicata or P. geniculate. The Hordeum (H genome)-like sequences of E. trachycaulus are polyphyletic in the Pepc tree, suggesting that the H genome in E. trachycaulus was contributed by multiple sources, whether due to multiple origins or introgression resulting from subsequent hybridization. Failure to recovering St copy of Pepc sequence in most accessions of E. trachycaulus might be caused by genome convergent evolution in allopolyploids. Multiple copies of H-like Pepc sequence from each accession with relative large deletions and insertions might be caused by either instability of Pepc sequence in H- genome or incomplete concerted evolution. Our results highlighted complex evolutionary history of E. trachycaulus.

Comparison of Acetyl-CoA carboxylase 1 (Acc-1) gene diversity among different Triticeae genomes.

It has widely been documented that life form and mating system have significant influences on genetic diversity. In the tribe Triticeae, several genera contain both annual and perennial species, whereas other genera comprise strictly annual or perennial species. It was suggested that Triticeae annuals have originated from Triticeae perennials. The present study aims to analyze nucleotide diversity of Acc-1 gene among different Triticeae genomes, and attempts to link effects of life history (annuals and perennials) and mating systems. The nucleotide diversity of 364 Acc-1 sequences in Triticeae species was characterized. The highest estimates of nucleotide diversity values (π=0.01919, θ=0.03515) were found for the Ns genome among the genomes analyzed. Nucleotide diversities in the D genome and Ns genome of polyploids are higher than those in respective genomes of diploids, while in the St genome of polyploids, it is lower than that in the St genome of diploids. The averaged π value (0.013705) in the genomes of perennials is more than twice of the value (0.00508) in the genomes of annuals. The averaged π value (0.01323) in the genomes of outcrossing species is two-fold of the value (0.005664) in the genomes of selfer. Our results suggested that the evolutionary history and mating system may play an important role in determining nucleotide diversity of Acc-1 gene in each genome.