Expression patterns of microRNAs in different organs and developmental stages of a superhybrid rice LYP9 and its parental lines

Heterosis is an important phenomenon, and the molecular mechanisms underlying heterosis are still enigmatic. microRNAs (miRNAs) play vital roles in many aspects of plant development. A set of miRNAs was selected to investigate the roles of miRNAs in heterosis displayed in a superhybrid rice. We analysed the expression patterns of miRNAs in different organs and developmental stages of the superhybrid rice and its parental lines. All possible modes of miRNA action were observed, including additive, high‐ and low‐parent value, above high‐ and below low‐parent value. Different organs and developmental stages exhibited different modes of miRNA expression. Overall, the non‐additive mode is the predominant expression pattern of miRNAs observed in this superhybrid. Many heterotic QTL intervals harbour miRNAs, whose expression patterns reveal their specific roles in different organs and developmental stages. miRNAs regulate the expression levels of target genes that have important functions in plant development. The predominant non‐additive mode of miRNA expression pattern in the hybrid suggests that miRNAs play critical roles in hybrid development, in particular, those miRNAs located in the heterotic QTL intervals may have important roles in heterosis. Our research sheds new light on understanding of the molecular mechanisms of heterosis.     

玉米雌穗发育期基因差异表达与杂种优势的研究

杂种优势在提高粮食作物特别是玉米的产量方面具有重要的作用。然而,杂种优势的原理却仍然是一个世界性的难题。用12个玉米自交系及其按不完全双列杂交组配的33个杂交种为材料,分4个不同发育时期取杂交种及其亲本的雌穗组织,利用差异显示技术,分析杂种与亲本的基因差异表达类型及其与7个主要农艺性状的杂种表现和杂种优势的相关关系。发现1):在5种表达类型中单态表达(基因在杂交种和双亲中同时表达的类型)的数量最大,这说明杂种优势的形成不仅与基因的表达与否相关,还与大量基因的上调或下调表达相关;2):在玉米雌幼穗的发育初期杂交种与双亲的基因表达差异最大,这可能与雌穗发育初期器官的形成和发育相关,因此这一时期差异表达(在质的方面)的基因对产量性状和杂种优势的形成具有密切关系;3):综合各种基因表达类型与产量性状和杂种优势的关系,发现某些基因在杂种中的沉默表达可以促进籽粒的发育和抑制幼穗中小花的发育。     

Deep Sequencing of Maize Small RNAs Reveals a Diverse Set of MicroRNA in Dry and Imbibed Seeds

Seed germination plays a pivotal role during the life cycle of plants. As dry seeds imbibe water, the resumption of energy metabolism and cellular repair occur and miRNA-mediated gene expression regulation is involved in the reactivation events. This research was aimed at understanding the role of miRNA in the molecular control during seed imbibition process. Small RNA libraries constructed from dry and imbibed maize seed embryos were sequenced using the Illumina platform. Twenty-four conserved miRNA families were identified in both libraries. Sixteen of them showed significant expression differences between dry and imbibed seeds. Twelve miRNA families, miR156, miR159, miR164, miR166, miR167, miR168, miR169, miR172, miR319, miR393, miR394 and miR397, were significantly down-regulated; while four families, miR398, miR408, miR528 and miR529, were significantly up-regulated in imbibed seeds compared to that in dry seeds. Furthermore, putative novel maize miRNAs and their target genes were predicted. Target gene GO analysis was performed for novel miRNAs that were sequenced more than 50 times in the normalized libraries. The result showed that carbohydrate catabolic related genes were specifically enriched in the dry seed, while in imbibed seed target gene enrichment covered a broad range of functional categories including genes in amino acid biosynthesis, isomerase activity, ligase activity and others. The sequencing results were partially validated by quantitative RT-PCR for both conserved and novel miRNAs and the predicted target genes. Our data suggested that diverse and complex miRNAs are involved in the seed imbibition process. That miRNA are involved in plant hormone regulation may play important roles during the dry-imbibed seed transition.     

Cloning and characterization of miRNAs from maize seedling roots under low phosphorus stress

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition in plants and animals. In this study, a small RNA library was constructed to identify conserved miRNAs as well as novel miRNAs in maize seedling roots under low level phosphorus stress. Twelve miRNAs were identified by high throughput sequencing of the library and subsequent analysis, two belong to conserved miRNA families (miRNA399b and miRNA156), and the remaining ten are novel and one of latter is conserved in gramineous species. Based on sequence homology, we predicted 125 potential target genes of these miRNAs and then expression patterns of 7 miRNAs were validated by semi-RT-PCR analysis. MiRNA399b, Zma-miR3, and their target genes (Zmpt1 and Zmpt2) were analyzed by real-time PCR. It is shown that both miRNA399b and Zma-miR3 are induced by low phosphorus stress and regulated by their target genes (Zmpt1 and Zmpt2). Moreover, Zma-miR3, regulated by two maize inorganic phosphate transporters as a newly identified miRNAs, would likely be directly involved in phosphate homeostasis, so was miRNA399b in Arabidopsis and rice. These results indicate that both conserved and maize-specific miRNAs play important roles in stress responses and other physiological processes correlated with phosphate starvation, regulated by their target genes. Identification of these differentially expressed miRNAs will facilitate us to uncover the molecular mechanisms underlying the progression of maize seedling roots development under low level phosphorus stress.     

Spatial and temporal expression modes of MicroRNAs in an elite rice hybrid and its parental lines

Heterosis is a commonly observed phenomenon in nature and refers to the superior performance of hybrids relative to both parents. The molecular mechanisms of heterosis are mostly unknown. Quantitative trait locus (QTL) mapping has been used to explain the genetic basis of heterosis, and large amounts of QTLs have been mapped for various agronomic traits, but the nature of QTL contributing to heterosis is still enigmatic. MicroRNAs (miRNAs) are master regulators in the processes of plant development and trait performance, and many miRNAs are predicted to reside in QTL intervals. We analyzed the expression modes of miRNAs, which were picked up from miRNA databases and chosen from those predicted from QTL intervals by bioinformatic approaches, in different organs at developmental stages of an elite rice hybrid and its parents. All possible modes of action for miRNA expression were detected, but most miRNAs showed nonadditive mode, and different stages and distinct organs displayed different patterns of miRNA expression. A large proportion of miRNAs were not detected for expression in leaves but expressed in the culms and roots of the hybrid at tillering stage. MiRNAs from grain-weight QTL intervals have multiple effects on grain development. Together, our results reveal that miRNAs, especially those from QTL intervals, play roles in heterotic performance in this elite rice hybrid, our results also shade new light on understanding the molecular mechanisms of heterosis.     

Genome‐wide identification and characterization of microRNAs responding to ABA and GA in maize embryos during seed germination

• MicroRNAs (miRNAs) are an important class of non‐coding small RNAs that regulate the expression of target genes through mRNA cleavage or translational inhibition. Previous studies have revealed their roles in regulating seed dormancy and germination in model plants such as Arabidopsis thaliana, rice (Oryza sativa) and maize (Zea mays). However, the miRNA response to exogenous gibberellic acid (GA) and abscisic acid (ABA) during seed germination in maize has yet to be explored.
• In this study, small RNA libraries were generated and sequenced from maize embryos treated with GA, ABA or double‐distilled water as control.
• A total of 247 miRNAs (104 known and 143 novel) were identified, of which 45 known and 53 novel miRNAs were differentially expressed in embryos in the different treatment groups. In total, 74 (37 up‐regulated and 37 down‐regulated) and 55 (23 up‐regulated and 32 down‐regulated) miRNAs were expressed in response to GA and to ABA, respectively, and a total of 18 known and 38 novel miRNAs displayed differential expression between the GA‐ and ABA‐treated groups. Using bioinformatics tools, we predicted the target genes of the differentially expressed miRNAs. Using GO enrichment and KEGG pathway analysis of these targets, we showed that miRNAs differentially expressed in our samples affect genes encoding proteins involved in the peroxisome, ribosome and plant hormonal signalling pathways.
• Our results indicate that miRNA‐mediated gene expression influences the GA and ABA signalling pathways during seed germination.

     

Heterosis associated gene expression in maize embryos 6 days after fertilization exhibits additive,dominant and overdominant pattern

Heterosis is important for conventional plant breeding and is intensively used to increase the productivity of crop plants. Genetic processes shortly after fertilization might be of particular importance with respect to heterosis, because coordination of the diverse genomes establishes a basis for future performance of the sporophyte. Here we demonstrate a strong crossbreeding advantage of hybrid maize embryos as early as 6 days after fertilization in a modern maize hybrid and provide the first embryo specific analysis of associated gene expression pattern at this early stage of development. We identified differentially expressed genes between hybrid embryos and the parental genotypes by a combined approach of suppression subtractive hybridization and differential screening by microarray hybridizations. Association of heterosis in embryos with genes related to signal transduction and other regulatory processes was implied by the enrichment of these functional classes among the identified gene set. Quantitative RT-PCR analysis validated the expression pattern of 7 of 12 genes analysed and revealed predominantly additive, but also dominant and overdominant expression patterns in hybrid embryos. These patterns indicate that gene regulatory interactions among parental alleles act at this early developmental stage and the genes identified provide entry points for the exploration of gene regulatory networks associated with the specification of the phenomenon heterosis in the plant life cycle.     

玉米microRNAs及其靶基因的生物信息学预测

microRNAs (miRNAs) 是一类非编码的小分子RNA, 通过碱基互补调控靶基因的表达。鉴定和发现新的miRNAs及其靶基因, 对揭示miRNAs在基因表达调控中的作用至关重要。玉米全基因组测序工作开展较晚, 已经鉴定登记的miRNAs很少, 对靶基因的调控作用尚待解明。文章根据miRNA进化上的保守性, 以已知的植物miRNAs为探针, 与相关数据库中玉米表达序列标签(EST)和基因组序列(GSS)中的非编码序列比对, 共发现11个新的miRNA前体。虽然在序列长度和二级结构方面各有变化, 但这11个前体均可折叠形成miRNA家族的标准二级结构。通过靶基因预测, 找到其中7条miRNAs的26个靶基因, 分别编码与新陈代谢、信号转导、转录调节、跨膜运输、生物和非生物胁迫及叶绿体组装等相关的蛋白。这些miRNAs及其靶基因的鉴定, 补充了miRNA数据库的不足。     

利用深度测序技术检测玉米根系和叶片中已知的microRNAs

microRNA(miRNA)是一类具有20～24nt核苷酸长度的非蛋白质编码的内源小分子RNA,它在植物生长发育和逆境胁迫响应等过程中发挥着重要作用。文章利用基于Illumina/Solexa原理的小分子RNA深度测序技术,结合生物信息学的方法对玉米根系和叶片中已知miRNA的类型、丰度及靶基因进行了分析。研究发现,在根系中共检测到92个已知的miRNA,分别属于18个miRNA家族,其表达丰度在1～105943之间;在叶片中,共发现86个已知的miRNA,分别属于17个miRNA家族,其表达丰度在1～85973之间。靶基因预测结果表明,根系中的18个miRNA家族共靶向54个蛋白,进一步的功能预测发现,这些基因涉及了转录调控、物质能量代谢、电子传递、胁迫响应和信号转导等过程。以上研究结果表明,就已知的miRNA而言,无论是miRNA的类型还是表达丰度,在玉米根系和叶片中都存在较大差异。     

Identification and characterization of a repertoire of genes differentially expressed in developing top ear shoots between a superior hybrid and its parental inbreds in Zea mays L.

Heterosis has been widely used in crop breeding and production; however, little is known about the genes controlling trait heterosis. The shortage of genes known to function in heterosis significantly limits our understanding of the molecular basis underlying heterosis. Here, we report 748 genes differentially expressed (DG) in the developing top ear shoots between a maize heterotic F₁ hybrid (Mo17 × B73) and its parental inbreds identified using maize microarrays containing 28,608 unigene features. Of the 748 DG, over 600 were new for the inbred and hybrid combination. The DG were enriched for 35 of the total 213 maize gene ontology (GO) terms, including those describing photosynthesis, respiration, DNA replication, metabolism, and hormone biosynthesis. From the DG, we identified six genes involved in glycolysis, three genes in the citrate cycle, and four genes in the C4-dicarboxylic acid cycle. We mapped 533 of the 748 DG to the maize B73 genome, 298 (55.9 %) of which mapped to the QTL intervals of 11 maize ear traits. Moreover, we compared the repertoire of the DG with that of 14-day seedlings of the same inbred and hybrid combination. Only approximately 5 % of the DG was shared between the two organs and developmental stages. Furthermore, we mapped 417 (55.7 %) of the 748 maize DG to the QTL intervals of 26 rice yield-related traits. Therefore, this study provides a repertoire of genes useful for identification of genes involved in maize ear trait heterosis and information for a better understanding of the molecular basis underlying heterosis in maize.     

水稻杂种一代与亲本幼苗基因表达差异的分析

杂种优势是一种普遍存在的生物学现象,其形成的原因十分复杂。本世纪初,Bruce和Shull相继提出的杂种优势形成的显性互补假设和超亲优势假设至今仍作为一种理论模型而缺乏实验证实。水稻杂种优势的利用自70年代三系配套技术建立得到了广泛的应用,但水稻杂种优势形成的遗传学基础目前还知之甚少。在水稻杂种优势形成机理研究中,分别从生理生化代谢、同工酶分析、DNA限制性片段多态性和DNA含量差异进行了分析,但杂种优势形成的分子机理仍未得到阐明。杂种优势的形成是与异质化相关的过程,它涉及到两个遗传背景不同的体系的相互作用。因此,在相互作用过程中,亲本基因的表达与调控就决定了杂种一代的基因表达类型和特性。因此,我们从分析基因表达与调控入手,运用mRNA差异展示技术分析了玉米杂种一代与亲本基因表达的差异,揭示了不少有意义的现象。本研究以水稻籼型杂交组合(汕优63:珍汕97A×明恢63)为材料,探讨水稻杂种一代与亲本基因表达的差异,揭示了杂种优势形成过程中的一些重要现象。