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.     

MicroRNA transcriptomic analysis of heterosis during maize seed germination

Heterosis has been utilized widely in the breeding of maize and other crops, and plays an important role in increasing yield, improving quality and enhancing stresses resistance, but the molecular mechanism responsible for heterosis is far from clear. To illustrate whether miRNA-dependent gene regulation is responsible for heterosis during maize germination, a deep-sequencing technique was applied to germinating embryos of a maize hybrid, Yuyu22, which is cultivated widely in China and its parental inbred lines, Yu87-1 and Zong3. The target genes of several miRNAs showing significant expression in the hybrid and parental lines were predicted and tested using real-time PCR. A total of 107 conserved maize miRNAs were co-detected in the hybrid and parental lines. Most of these miRNAs were expressed non-additively in the hybrid compared to its parental lines. These results indicated that miRNAs might participate in heterosis during maize germination and exert an influence via the decay of their target genes. Novel miRNAs were predicted follow a rigorous criterion and only the miRNAs detected in all three samples were treated as a novel maize miRNA. In total, 34 miRNAs belonged to 20 miRNA families were predicted in germinating maize seeds. Global repression of miRNAs in the hybrid, which might result in enhanced gene expression, might be one reason why the hybrid showed higher embryo germination vigor compared to its parental lines.     

Interacted QTL Mapping in Partial NCII Design Provides Evidences for Breeding by Design

The utilization of heterosis in rice, maize and rapeseed has revolutionized crop production. Although elite hybrid cultivars are mainly derived from the F₁ crosses between two groups of parents, named NCII mating design, little has been known about the methodology of how interacted effects influence quantitative trait performance in the population. To bridge genetic analysis with hybrid breeding, here we integrated an interacted QTL mapping approach with breeding by design in partial NCII mating design. All the potential main and interacted effects were included in one full model. If the number of the effects is huge, bulked segregant analysis were used to test which effects were associated with the trait. All the selected effects were further shrunk by empirical Bayesian, so significant effects could be identified. A series of Monte Carlo simulations was performed to validate the new method. Furthermore, all the significant effects were used to calculate genotypic values of all the missing F₁ hybrids, and all these F₁ phenotypic or genotypic values were used to predict elite parents and parental combinations. Finally, the new method was adopted to dissect the genetic foundation of oil content in 441 rapeseed parents and 284 F₁ hybrids. As a result, 8 main-effect QTL and 37 interacted QTL were found and used to predict 10 elite restorer lines, 10 elite sterile lines and 10 elite parental crosses. Similar results across various methods and in previous studies and a high correlation coefficient (0.76) between the predicted and observed phenotypes validated the proposed method in this study.     

Overdominant epistatic loci are the primary genetic basis of inbreeding depression and heterosis in rice. I. Biomass and grain yield

To understand the genetic basis of inbreeding depression and heterosis in rice, main-effect and epistatic QTL associated with inbreeding depression and heterosis for grain yield and biomass in five related rice mapping populations were investigated using a complete RFLP linkage map of 182 markers, replicated phenotyping experiments, and the mixed model approach. The mapping populations included 254 F(10) recombinant inbred lines derived from a cross between Lemont (japonica) and Teqing (indica) and two BC and two testcross hybrid populations derived from crosses between the RILs and their parents plus two testers (Zhong 413 and IR64). For both BY and GY, there was significant inbreeding depression detected in the RI population and a high level of heterosis in each of the BC and testcross hybrid populations. The mean performance of the BC or testcross hybrids was largely determined by their heterosis measurements. The hybrid breakdown (part of inbreeding depression) values of individual RILs were negatively associated with the heterosis measurements of their BC or testcross hybrids, indicating the partial genetic overlap of genes causing hybrid breakdown and heterosis in rice. A large number of epistatic QTL pairs and a few main-effect QTL were identified, which were responsible for >65% of the phenotypic variation of BY and GY in each of the populations with the former explaining a much greater portion of the variation. Two conclusions concerning the loci associated with inbreeding depression and heterosis in rice were reached from our results. First, most QTL associated with inbreeding depression and heterosis in rice appeared to be involved in epistasis. Second, most ( approximately 90%) QTL contributing to heterosis appeared to be overdominant. These observations tend to implicate epistasis and overdominance, rather than dominance, as the major genetic basis of heterosis in rice. The implications of our results in rice evolution and improvement are discussed.     

Molecular marker heterozygosity and hybrid performance in indica and japonica rice

An essential assumption underlying markerbased prediction of hybrid performance is a strong linear correlation between molecular marker heterozygosity and hybrid performance or heterosis. This study was intended to investigate the extent of the correlations between molecular marker heterozygosity and hybrid performance in crosses involving two sets of rice materials, 9 indica and 11 japonica varieties. These materials represent a broad spectrum of the cultivated rice gene pool including landraces, primitive cultivars, historically important cultivars, modern elite cultivars and parents of superior hybrids. Varieties within each set were intermated in all possible nonreciprocal pairs resulting in 36 crosses in the indica set and 55 in the japonica set. The F₁s and their parents, 111 entries in total, were examined for performance of seven traits in a replicated field trial. The parents were surveyed for polymorphisms using 96 RFLP and ten SSR markers selected at regular intervals from a published molecular marker linkage map. Molecular marker genotypes of the F₁ hybrids were deduced from the parental genotypes. The analysis showed that, with very few exceptions, correlations in the indica dataset were higher than in that of their japonica counterparts. Among the seven traits analyzed, plant height showed the highest correlation between heterozygosity and hybrid performance and heteorsis in both indica and japonica datasets. Correlations were low to intermediate between hybrid performance and heterozygosity (both general and specific) in yield and yield component traits in both indica and japonica sets, and also low to intermediate between specific heterozygosity and heterosis in the indica set, whereas very little correlation was detected between heterosis and heterozygosity (either general or specific) in the japonica set. In comparison to the results from our previous studies, we concluded that the relationship between molecular marker heterozygosity and heterosis is variable, depending on the genetic materials used in the study, the diversity of rice germplasms and the complexity of the genetic basis of heterosis.     

Identification of Conserved and Novel MicroRNAs in the Pacific Oyster Crassostrea gigas by Deep Sequencing

MicroRNAs (miRNAs) play important roles in regulatory processes in various organisms. To date many studies have been performed in the investigation of miRNAs of numerous bilaterians, but limited numbers of miRNAs have been identified in the few species belonging to the clade Lophotrochozoa. In the current study, deep sequencing was conducted to identify the miRNAs of Crassostrea gigas (Lophotrochozoa) at a genomic scale, using 21 libraries that included different developmental stages and adult organs. A total of 100 hairpin precursor loci were predicted to encode miRNAs. Of these, 19 precursors (pre-miRNA) were novel in the oyster. As many as 53 (53%) miRNAs were distributed in clusters and 49 (49%) precursors were intragenic, which suggests two important biogenetic sources of miRNAs. Different developmental stages were characterized with specific miRNA expression patterns that highlighted regulatory variation along a temporal axis. Conserved miRNAs were expressed universally throughout different stages and organs, whereas novel miRNAs tended to be more specific and may be related to the determination of the novel body plan. Furthermore, we developed an index named the miRNA profile age index (miRPAI) to integrate the evolutionary age and expression levels of miRNAs during a particular developmental stage. We found that the swimming stages were characterized by the youngest miRPAIs. Indeed, the large-scale expression of novel miRNAs indicated the importance of these stages during development, particularly from organogenetic and evolutionary perspectives. Some potentially important miRNAs were identified for further study through significant changes between expression patterns in different developmental events, such as metamorphosis. This study broadened the knowledge of miRNAs in animals and indicated the presence of sophisticated miRNA regulatory networks related to the biological processes in lophotrochozoans.     

Identification of novel MiRNAs and MiRNA expression profiling during grain development in indica rice

ABSTRACT: BACKGROUND: MicroRNAs (miRNAs) modulate gene expression in different tissues and at diverse developmental stages, including grain development in japonica rice. To identify novel miRNAs in indica rice and to study their expression patterns during the entire grain filling process, small RNAs from all stages of grain development were sequenced and their expression patterns were studied using customized miRNA chips. RESULTS: A total of 21 conserved and 91 non-conserved miRNA families were found in developing indica grains. We also discovered 11 potential novel miRNAs based on the presence of their miRNA*s. Expression patterns of these identified miRNAs were analyzed using customized miRNA chips. The results showed that during the filling phase about half of the detected miRNAs were up-regulated, whereas the remainder were down-regulated. Predicted targets of differentially expressed miRNAs may participate in carbohydrate metabolism, hormone signaling and pathways associated with seed maturity, suggesting potentially important roles in rice grain development. CONCLUSIONS: This study is the first genome-wide investigation of miRNAs during the grain-filling phase of an indica variety of rice. The novel miRNAs identified might be involved in new miRNA regulatory pathways for grain development. The complexity of these miRNAs and their targets and interactions require further study to obtain a better understanding of the molecular mechanisms underlying grain development. Key words: miRNA, grain filling, indica rice.     

Expression analysis of miRNAs and highly-expressed small RNAs in two rice subspecies and their reciprocal hybrids

Heterosis,or hybrid vigor,is the phenomenon whereby progeny of two inbred lines exhibit superior agronomic performance compared with either parent.We analyzed the expression of miRNAs and highly expressed small RNAs(defined according to Solexa sequencing results)in two rice(Oryza sativa)subspecies(japonica cv.Nipponbare and indica cv.93-11)and their reciprocal hybrids using microarrays.We found that of all the 1141 small RNAs tested,140(12%,140 of 1141)and 157(13%,157 of 1141)were identified being significantly differentially expressed in two reciprocal hybrids,respectively.All possible modes of action,including additive,high- and low- parent,above high- and below low-parent modes were exhibited.Both F1 hybrids showed non-additive expression patterns,with downregulation predominating.Interestingly,15 miRNAs displayed stark opposite expression trends relative to midparent in reciprocal hybrids.Computational prediction of targets of differentially expressed miRNAs showed that they participated in multifaceted developmental pathways,and were not distinguishable from the targets of non-differentially expressed miRNAs.Together,our findings reveal that small RNAs play roles in heterosis and add a new layer in the understanding and exploitation of molecular mechanisms of heterosis.     

组学技术揭示水稻杂种优势遗传机制

杂种优势是杂交后代在生长或生殖性状上表现出优于亲本的现象。虽然杂种优势在农业生产上已广为应用,但其分子机理仍不清楚。最近,中国科学家通过分析17个代表性杂交稻(Oryza sativa)品种,共10 074个F2个体的全基因组序列和表型,对水稻产量杂种优势相关位点进行了系统定位和解析。此外,中国另一个科研小组通过整合杂交稻亲本和杂交种的表型组、转录组及基因组等多层次数据,深入研究了超级杂交稻两优培九产量的杂种优势基础。这些研究不仅为杂种优势理论的建立提供了新数据,也为水稻育种实践提供了有益的指导。     

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.     

Identification and Expression Analysis of microRNAs at the Grain Filling Stage in Rice(Oryza sativa L.)via Deep Sequencing

MicroRNAs (miRNAs) have been shown to play crucial roles in the regulation of plant development. In this study, high-throughput RNA-sequencing technology was used to identify novel miRNAs, and to reveal miRNAs expression patterns at different developmental stages during rice (Oryza sativa L.) grain filling. A total of 434 known miRNAs (380, 402, 390 and 392 at 5, 7, 12 and 17 days after fertilization, respectively.) were obtained from rice grain. The expression profiles of these identified miRNAs were analyzed and the results showed that 161 known miRNAs were differentially expressed during grain development, a high proportion of which were up-regulated from 5 to 7 days after fertilization. In addition, sixty novel miRNAs were identified, and five of these were further validated experimentally. Additional analysis showed that the predicted targets of the differentially expressed miRNAs may participate in signal transduction, carbohydrate and nitrogen metabolism, the response to stimuli and epigenetic regulation. In this study, differences were revealed in the composition and expression profiles of miRNAs among individual developmental stages during the rice grain filling process, and miRNA editing events were also observed, analyzed and validated during this process. The results provide novel insight into the dynamic profiles of miRNAs in developing rice grain and contribute to the understanding of the regulatory roles of miRNAs in grain filling.     

Assembly of yield heterosis of an elite rice hybrid is promising by manipulating dominant quantitative trait loci

In the past, rice hybrids with strong heterosis have been obtained empirically, by developing and testing thousands of combinations. Here, we aimed to determine whether heterosis of an elite hybrid could be achieved by manipulating major quantitative trait loci. We used 202 chromosome segment substitution lines from the elite hybrid Shanyou 63 to evaluate single segment heterosis (SSH) of yield per plant and identify heterotic loci. All nine detected heterotic loci acted in a dominant fashion, and no SSH exhibited overdominance. Functional alleles of key yield-related genes Ghd7, Ghd7.1, Hd1, and GS3 were dispersed in both parents. No functional alleles of three investigated genes were expressed at higher levels in the hybrids than in the more desirable parents. A hybrid pyramiding eight heterotic loci in the female parent Zhenshan 97 background had a comparable yield to Shanyou 63 and much higher yield than Zhenshan 97. Five hybrids pyramiding eight or nine heterotic loci in the combined parental genome background showed similar yield performance to that of Shanyou 63. These results suggest that dominance underlying functional complementation is an important contributor to yield heterosis and that heterosis assembly might be successfully promised by manipulating several major dominant heterotic loci.     

野生稻高产QTL高效表达的光合生理基础

以携带野生稻高产QTL的晚稻新恢复系远恢611所配部分强优势组合为材料,对其部分光合生理指标进行测定的结果表明,远恢611系列组合杂种优势强,穗大粒多,库容量大,具有超高产潜力;后期上面3片功能叶宽大、直立、叶面积大,与茎秆夹角小,不披垂;比叶重大而稳定,不早衰;剑叶净光合速率高,库很大且源较足是远恢611系列组合高产的主要生理原因;也可能是野生稻高产QTL高效表达的重要生理基础。     

Early changes of gene activity in developing seedlings of Arabidopsis hybrids relative to parents may contribute to hybrid vigour

Hybrid vigour (heterosis) has been used for decades in crop industries, especially in the production of maize and rice. Hybrid varieties usually exceed their parents in plant biomass and seed yield. But the molecular basis of hybrid vigour is not fully understood. In this project, we studied heterosis at early stages of seedling development in Arabidopsis hybrids derived from crossing Ler and C24 accessions. We found that early heterosis is associated with non‐additive gene expression that resulted from earlier changes in gene expression in the hybrids relative to the parents. The non‐additively expressed genes are involved in metabolic pathways, including photosynthesis, critical for plant growth. The early increased expression levels of genes involved in energy production in hybrids is associated with heterosis in the young seedlings that could be essential for biomass heterosis at later developmental stages of the plant.     

MicroRNA expression changes following synthesis of three full-sib <Emphasis Type="Italic">Populus</Emphasis> triploid populations with different heterozygosities

Key message

Through high-throughput sequencing, we compared the relative expression levels of miRNA in three full-sib Populus triploid populations with that in their parents and one diploid hybrid population. We found similar numbers of miRNAs differentially expressed between the parents and the four progeny hybrid populations. In addition, unbalanced parental expression level dominance of miRNAs were found in the three allotriploid and interspecific hybrid populations, which may reprogram gene expression networks and contribute to the growth of Populus hybrids. These results indicated that hybridization has a great impact on the miRNA expression variation in the newly synthesized Populus triploid and diploid hybrid populations. However, we also found no significant differences in miRNA expression among one diploid and three triploid hybrid populations, hinting that miRNA abundances do not increase with the genome content. No dosage effect of miRNA expression could lead to dosage-dependent negative effects on target genes and their downstream pathway in polyploids. We speculate that polyploids may gain advantages from the slight decrease in miRNA regulation, suggesting an important molecular mechanism of polyploid advantage.

Abstract

Hybridization with three types of induced 2n gametes transmitted different parental heterozygosities has been proven as an efficient method for Populus triploid production. Several researches have shown that miRNA could be non-additively expressed in allopolyploids. However, it is still unclear whether the non-additively expressed miRNAs result from the effect of hybridization or polyploidization, and whether a dose response to the additional genomic content exists for the expression of miRNA. Toward this end, through high-throughput sequencing, we compared the expression levels of miRNA in three full-sib Populus triploid populations with that in their parents and one interspecific hybrid population. We found similar numbers of miRNAs differentially expressed between the parents and the four progeny hybrid populations. Unbalanced parental expression level dominance of miRNAs were found in the three triploid and diploid hybrid populations, which may reprogram gene expression networks and affect the growth of Populus hybrids. These results indicated that hybridization has a great impact on the miRNA expression variation in the newly synthesized Populus triploid and diploid hybrid populations. However, we also found no significant differences in miRNA expression among the three triploid populations and the diploid hybrid population. No dosage effect of miRNA expression could lead to dosage-dependent negative effects on target genes and their downstream pathway in polyploids. We speculate that polyploids may gain advantages from the decrease in miRNA negative regulation, suggesting an important molecular mechanism of polyploid advantage.

     

Comparative proteomic study reveals dynamic proteome changes between superhybrid rice LYP9 and its parents at different developmental stages

Heterosis is a common phenomenon in which the hybrids exhibit superior agronomic performance than either inbred parental lines. Although hybrid rice is one of the most successful apotheoses in crops utilizing heterosis, the molecular mechanisms underlying rice heterosis remain elusive. To gain a better understanding of the molecular mechanisms of rice heterosis, comparative leaf proteomic analysis between a superhybrid rice LYP9 and its parental cultivars 9311 and PA64s at tillering, flowering and grain-filling stages were carried out. A total of 384 differentially expressed proteins (DP) were detected and 297 DP were identified, corresponding to 222 unique proteins. As DP were divided into those between the parents (DP_PP) and between the hybrid and its parents (DP_HP), the comparative results demonstrate that proteins in the categories of photosynthesis, glycolysis, and disease/defense were mainly enriched in DP. Moreover, the number of identified DP_HP involved in photosynthesis, glycolysis, and disease/defense increased at flowering and grain-filling stages as compared to that at the tillering stage. Most of the up-regulated DP_HP involved in the three categories showed greater expression in LYP9 at flowering and grain-filling stages than at the tillering stage. In addition, CO₂ assimilation rate and apparent quantum yield of photosynthesis also showed a greater increase in LYP9 at flowering and grain-filling stages than at the tillering stage. These results suggest that the proteins involved in photosynthesis, glycolysis, and disease/defense as well as their dynamic regulation at different developmental stages may be responsible for heterosis in rice.     

Comparison of quantitative trait loci controlling seedling characteristics at two seedling stages using rice recombinant inbred lines

A better understanding of the genetics of seedling characteristics in rice could be helpful in improving rice varieties. Zhenshan 97 and Minghui 63, the parents of Shanyou 63, an elite hybrid developed during the last decade in China, vary greatly with respect to their physiological and morphological traits at the seedling growth stage. In this study, we used a population of 240 recombinant inbred lines derived from a cross between Zhenshan 97 and Minghui 63 to identify quantitative trait loci (QTL) for seedling characteristics. All plant material was grown in hydroponic culture. Data for the following characters were collected at 30 days and 40 days post-sowing: plant height, shoot dry matter weight (SDW), maximum root length, root dry weight (RDW), total dry weight , and root-shoot ratio (the ratio of SDW to RDW). Analysis using composite interval mapping detected 16 QTL for the six traits in 30-day-old seedlings. Of these 16 QTL, Minghui 63 alleles increased trait values at only two of them. The QTL in the vicinity of R3166 on chromosome 5 simultaneously influenced PH, SDW, MRL, RDW, and TDW in the same direction. Twenty QTL were detected for the same traits in the 40-day-old seedlings. However, at this stage Minghui 63 alleles increased trait values at eight QTL. The QTL linked to R3166 also affected PH, SDW, MRL, RDW, and TDW. Only four QTL were common to the two stages. These results clearly indicate that different genes (QTL) control the same traits during different time intervals. Zhenshan 97 alleles had positive effects during the first 30 days of seedling growth, but thereafter the positive effects of Minghui 63 alleles on seedling growth gradually became more pronounced.