The conservation of allelic DNA methylation and its relationship with imprinting in maize.

Genomic imprinting refers to allele-specific expression of genes depending on parental origin, and it is regulated by epigenetic modifications. Intraspecific allelic variation for imprinting has been detected; however, the intraspecific genome-wide allelic epigenetic variation in maize and its correlation with imprinting variants remain unclear. Here, three reciprocal hybrids were generated by crossing Zea mays inbred lines CAU5, B73, and Mo17 in order to examine the intraspecific conservation of the imprinted genes in the kernel. The majority of imprinted genes exhibited intraspecific conservation, and these genes also exhibited interspecific conservation (rice, sorghum, and Arabidopsis) and were enriched in some specific pathways. By comparing intraspecific allelic DNA methylation in the endosperm, we found that nearly 15% of DNA methylation existed as allelic variants. The intraspecific whole-genome correlation between DNA methylation and imprinted genes indicated that DNA methylation variants play an important role in imprinting variants. Disruption of two conserved imprinted genes using CRISPR/Cas9 editing resulted in a smaller kernel phenotype. Our results shed light on the intraspecific correlation of DNA methylation variants and variation for imprinting in maize, and show that imprinted genes play an important role in kernel development.

Transcriptome-wide identification and characterization of genes exhibit allele-specific imprinting in maize embryo and endosperm.

BACKGROUND: Genomic imprinting refers to a subset of genes that are expressed from only one parental allele during seed development in plants. Studies on genomic imprinting have revealed that intraspecific variations in genomic imprinting expression exist in naturally genetic varieties. However, there have been few studies on the functional analysis of allele-specific imprinted genes. RESULTS: Here, we generated three reciprocal crosses among the B73, Mo17 and CAU5 inbred lines. Based on the transcriptome-wide analysis of allele-specific expression using RNA sequencing technology, 305 allele-specific imprinting genes (ASIGs) were identified in embryos, and 655 ASIGs were identified in endosperms from three maize F1 hybrids. Of these ASIGs, most did not show consistent maternal or paternal bias between the same tissue from different hybrids or different tissues from one hybrid cross. By gene ontology (GO) analysis, five and eight categories of GO exhibited significantly higher functional enrichments for ASIGs identified in embryo and endosperm, respectively. These functional categories indicated that ASIGs are involved in intercellular nutrient transport, signaling pathways, and transcriptional regulation of kernel development. Finally, the mutation and overexpression of one ASIG (Zm305) affected the length and width of the kernel. CONCLUSION: In this study, our data will be helpful in gaining further knowledge of genes exhibiting allele-specific imprinting patterns in seeds. The gain- and loss-of-function phenotypes of ASIGs associated with agronomically important seed traits provide compelling evidence for ASIGs as crucial targets to optimize seed traits in crop plants.

The Resistance of Maize to Ustilago maydis Infection Is Correlated with the Degree of Methyl Esterification of Pectin in the Cell Wall.

Common smut caused by Ustilago maydis is one of the dominant fungal diseases in plants. The resistance mechanism to U. maydis infection involving alterations in the cell wall is poorly studied. In this study, the resistant single segment substitution line (SSSL) R445 and its susceptible recurrent parent line Ye478 of maize were infected with U. maydis, and the changes in cell wall components and structure were studied at 0, 2, 4, 8, and 12 days postinfection. In R445 and Ye478, the contents of cellulose, hemicellulose, pectin, and lignin increased by varying degrees, and pectin methylesterase (PME) activity increased. The changes in hemicellulose and pectin in the cell wall after U. maydis infection were analyzed via immunolabeling using monoclonal antibodies against hemicellulsic xylans and high/low-methylated pectin. U. maydis infection altered methyl esterification of pectin, and the degree of methyl esterification was correlated with the resistance of maize to U. maydis. Furthermore, the relationship between methyl esterification of pectin and host resistance was validated using 15 maize inbred lines with different resistance levels. The results revealed that cell wall components, particularly pectin, were important factors affecting the colonization and propagation of U. maydis in maize, and methyl esterification of pectin played a role in the resistance of maize to U. maydis infection.

BSA-Seq and Transcriptomic Analysis Provide Candidate Genes Associated with Inflorescence Architecture and Kernel Orientation by Phytohormone Homeostasis in Maize.

The developmental plasticity of the maize inflorescence depends on meristems, which directly affect reproductive potential and yield. However, the molecular roles of upper floral meristem (UFM) and lower floral meristem (LFM) in inflorescence and kernel development have not been fully elucidated. In this study, we characterized the reversed kernel1 (rk1) novel mutant, which contains kernels with giant embryos but shows normal vegetative growth like the wild type (WT). Total RNA was extracted from the inflorescence at three stages for transcriptomic analysis. A total of 250.16-Gb clean reads were generated, and 26,248 unigenes were assembled and annotated. Gene ontology analyses of differentially expressed genes (DEGs) detected in the sexual organ formation stage revealed that cell differentiation, organ development, phytohormonal responses and carbohydrate metabolism were enriched. The DEGs associated with the regulation of phytohormone levels and signaling were mainly expressed, including auxin (IAA), jasmonic acid (JA), gibberellins (GA), and abscisic acid (ABA). The transcriptome, hormone evaluation and immunohistochemistry observation revealed that phytohormone homeostasis were affected in rk1. BSA-Seq and transcriptomic analysis also provide candidate genes to regulate UFM and LFM development. These results provide novel insights for understanding the regulatory mechanism of UFM and LFM development in maize and other plants.

Characterization of Genes That Exhibit Genotype-Dependent Allele-Specific Expression and Its Implications for the Development of Maize Kernel.

Heterosis or hybrid vigor refers to the superior phenotypic traits of hybrids relative to their parental inbred lines. An imbalance between the expression levels of two parental alleles in the F1 hybrid has been suggested as a mechanism of heterosis. Here, based on genome-wide allele-specific expression analysis using RNA sequencing technology, 1689 genes exhibiting genotype-dependent allele-specific expression (genotype-dependent ASEGs) were identified in the embryos, and 1390 genotype-dependent ASEGs in the endosperm, of three maize F1 hybrids. Of these ASEGs, most were consistent in different tissues from one hybrid cross, but nearly 50% showed allele-specific expression from some genotypes but not others. These genotype-dependent ASEGs were mostly enriched in metabolic pathways of substances and energy, including the tricarboxylic acid cycle, aerobic respiration, and energy derivation by oxidation of organic compounds and ADP binding. Mutation and overexpression of one ASEG affected kernel size, which indicates that these genotype-dependent ASEGs may make important contributions to kernel development. Finally, the allele-specific methylation pattern on genotype-dependent ASEGs indicated that DNA methylation plays a potential role in the regulation of allelic expression for some ASEGs. In this study, a detailed analysis of genotype-dependent ASEGs in the embryo and endosperm of three different maize F1 hybrids will provide an index of genes for future research on the genetic and molecular mechanism of heterosis.

Conservation Study of Imprinted Genes in Maize Triparental Heterozygotic Kernels.

Genomic imprinting is a classic epigenetic phenomenon related to the uniparental expression of genes. Imprinting variability exists in seeds and can contribute to observed parent-of-origin effects on seed development. Here, we conducted allelic expression of the embryo and endosperm from four crosses at 11 days after pollination (DAP). First, the F1 progeny of B73(♀) × Mo17(♂) and the inducer line CAU5 were used as parents to obtain reciprocal crosses of BM-C/C-BM. Additionally, the F1 progeny of Mo17(♀) × B73(♂) and CAU5 were used as parents to obtain reciprocal crosses of MB-C/C-MB. In total, 192 and 181 imprinted genes were identified in the BM-C/C-BM and MB-C/C-MB crosses, respectively. Then, by comparing the allelic expression of these imprinted genes in the reciprocal crosses of B73 and CAU5 (BC/CB), fifty-one Mo17-added non-conserved genes were identified as exhibiting imprinting variability. Fifty-one B73-added non-conserved genes were also identified by comparing the allelic expression of imprinted genes identified in BM-C/C-BM, MB-C/C-MB and MC/CM crosses. Specific Gene Ontology (GO) terms were not enriched in B73-added/Mo17-added non-conserved genes. Interestingly, the imprinting status of these genes was less conserved across other species. The cis-element distribution, tissue expression and subcellular location were similar between the B73-added/Mo17-added conserved and B73-added/Mo17-added non-conserved imprinted genes. Finally, genotypic and phenotypic analysis of one non-conserved gene showed that the mutation and overexpression of this gene may affect embryo and kernel size, which indicates that these non-conserved genes may also play an important role in kernel development. The findings of this study will be helpful for elucidating the imprinting mechanism of genes involved in maize kernel development.

Overexpression of Modified CENH3 in Maize Stock6-Derived Inducer Lines Can Effectively Improve Maternal Haploid Induction Rates.

Maize (Zea mays) doubled haploid (DH) breeding is a technology that can efficiently generate inbred lines with homozygous genetic backgrounds. Haploids are usually produced through in vivo induction by haploid inducer lines in maize. Currently, two approaches are usually used to develop maize haploid inducer lines. One is through the conventional breeding improvement based on the Stock6 germplasm, and this strategy is extensively used to induce maternal haploids in commercial maize DH breeding. Another strategy, newly developed but less utilized so far, is by genetic manipulation of the Centromeric Histone3 (CENH3) in regular lines. However, whether both approaches can be combined to develop the haploid inducer line with higher maternal haploid induction rate (HIR) has not been reported. In this study, we manipulated the Stock6-derived inducer lines by overexpressing maize CENH3 fused with different fluorescent protein tags and found that the engineered Stock6-derived lines showed an obvious increase in the maternal HIR. Intriguingly, this above strategy could be further improved by substituting a tail-altered CENH3 for the full-length CENH3 in the tagged expression cassette, resulting in a maternal HIR up to 16.3% that was increased by ~6.1% than Stock6-derived lines control. These results suggested that integration of two in vivo haploid induction methods could rapidly and effectively improve the maternal HIRs of maize Stock6-derived inducer lines, and provided a potentially feasible solution for further optimizing the process of commercial maize DH breeding.

Ectopic Expression of the Transcriptional Regulator silky3 Causes Pleiotropic Meristem and Sex Determination Defects in Maize Inflorescences.

Maize (Zea mays) is a monoecious plant, in which inflorescence morphogenesis involves complicated molecular regulatory mechanisms. Although many related genes have been cloned, our understanding of the molecular mechanism underlying maize inflorescence development remains limited. Here, we identified a maize semi-dominant mutant Silky3 (Si3), which displays pleiotropic defects during inflorescence development, including loss of determinacy and identity in meristems and floral organs, as well as the sexual transformation of tassel florets. We cloned the si3 gene using a map-based approach. Functional analysis reveals that SI3 is a nuclear protein and may act as a transcriptional regulator. Transcriptome analysis reveals that the ectopic expression of si3 strongly represses multiple biological processes, especially the flower development pathways. RNA in situ hybridization similarly shows that the expression patterns of genes responsible for flower development are changed in the Si3 mutant. In addition, the homeostasis of jasmonic acid and gibberellic acid are altered in the Si3 young tassels, and application of exogenous jasmonic acid can rescue the sex reversal phenotype of Si3 The defects we characterized in various regulatory pathways can explain the complex phenotypes of Si3 mutant, and this study deepens our knowledge of maize inflorescence development.

Sequential gene activation and gene imprinting during early embryo development in maize.

Gene imprinting is a widely observed epigenetic phenomenon in maize endosperm; however, whether it also occurs in the maize embryo remains controversial. Here, we used high-throughput RNA sequencing on laser capture microdissected and manually dissected maize embryos from reciprocal crosses between inbred lines B73 and Mo17 at six time points (3-13 days after pollination, DAP) to analyze allelic gene expression patterns. Co-expression analysis revealed sequential gene activation during maize embryo development. Gene imprinting was observed in maize embryos, and a greater number of imprinted genes were identified at early embryo stages. Sixty-four strongly imprinted genes were identified (at the threshold of 9:1) on manually dissected embryos 5-13 DAP (more imprinted genes at 5 DAP). Forty-one strongly imprinted genes were identified from laser capture microdissected embryos at 3 and 5 DAP (more imprinted genes at 3 DAP). Furthermore, of the 56 genes that were completely imprinted (at the threshold of 99:1), 36 were not previously identified as imprinted genes in endosperm or embryos. In situ hybridization demonstrated that most of the imprinted genes were expressed abundantly in maize embryonic tissue. Our results shed lights on early maize embryo development and provide evidence to support that gene imprinting occurs in maize embryos.

Single nucleus sequencing reveals spermatid chromosome fragmentation as a possible cause of maize haploid induction.

Production of maternal haploids using a conspecific haploid inducer is routine and highly efficient in maize. However, the underlying mechanism of haploid induction (HI) is unclear. We develop a method to isolate three nuclei from a pollen grain and four microspores from a tetrad for whole-genome sequencing. A high rate of aneuploidy is observed at the three-nucleus stage (6/22 pollens) rather than at the tetrad stage (1/72 microspores) in one HI line CAU5. Frequent aneuploidy is also observed in another two inducer lines, but not in two regular lines, which implies that HI may be associated with pollen aneuploidy. We further sequence the individual embryos and endosperms of 88 maize kernels crossing between regular and inducer lines. Genome-wide elimination of the CAU5-derived chromosome is identified in eight of 81 embryos. Together, these results suggest that continuous chromosome fragmentation occurring post meiosis in the gametophyte may cause haploidy of the embryo.

Strain, Negative Emotions, and Level of Criminality Among Chinese Incarcerated Women.

General strain theory (GST) has been one of the most frequently tested criminological theories. According to GST, strain tends to generate negative emotions, which create pressures for corrective action, such as crime and delinquency. Although GST has received strong empirical support, one under-addressed issue is the lack of diversity in sampling population in assessing the generalizability of the theory. Using survey data collected from 335 incarcerated women in four Chinese prisons, this study examined the impact of strain and negative emotions on the level of female criminality. The strain variable, physical abuse, and discrimination, exerted a positive effect on female inmates' levels of criminality, whereas negative emotions were not significantly related to female criminality. Two control variables, age of current offense and educational attainment, were predictive of female criminality, with younger and less-educated women having more serious criminality. Implications for future research and policy are discussed.

Phylogenetic analysis of Heliothis armigera cytoplasmic polyhedrosis virus type 14 and a series of dwarf segments found in the genome.

Full-length nucleotide sequences for the genome segments (S1-S6) of Heliothis armigera cytoplasmic polyhedrosis virus type 14 (HaCPV-14) have been characterized. Each segment consists of a single open reading frame with conserved motifs AGAA and AGCU at the 5' and 3' ends, respectively. Comparison of the proteins of HaCPV-14 with those of other members of the family Reoviridae suggests that S1 encodes an RNA-dependent RNA polymerase (RdRp), whilst S2 encodes a major capsid protein of the virus. Phylogenetic analysis of RdRps from 16 viruses in the family Reoviridae reveals that the genera Cypovirus and Oryzavirus may have originated from a common insect virus ancestor. A series of viable dwarf segments originating from S5 of HaCPV-14 has been identified. Analysis of the predicted secondary structures for these dwarf segments suggests that the signals essential for replication and packaging are located within the terminal sequences of these segments.