Identification of reliable QTLs and designed QTL breeding for grain shape and milling quality in the reciprocal introgression lines in rice.

Milling quality (MQ) and grain shape (GS) of rice (Oryza sativa L.) are correlated traits, both determine farmers' final profit. More than one population under multiple environments may provide valuable information for breeding selection on these MQ-GS correlations. However, suitable analytical methods for reciprocal introgression lines with linkage map for this kind of correlation remains unclear. In this study, our major tasks were (1) to provide a set of reciprocal introgression lines (composed of two BC2RIL populations) suitable for mapping by linkage mapping using markers/bins with physical positions; (2) to test the mapping effects of different methods by using MQ-GS correlation dissection as sample case; (3) to perform genetic and breeding simulation on pyramiding favorite alleles of QTLs for representative MQ-GS traits. Finally, with four analysis methods and data collected under five environments, we identified about 28.4 loci on average for MQ-GS traits. Notably, 52.3% of these loci were commonly detected by different methods and eight loci were novel. There were also nine regions harboring loci for different MQ-GS traits which may be underlying the MQ-GS correlations. Background independent (BI) loci were also found for each MQ and GS trait. All these information may provide useful resources for rice molecular breeding.

Heterodimer formed by ROC8 and ROC5 modulates leaf rolling in rice.

Moderately rolled leaf is one of the target traits of the ideal plant architecture in rice breeding. Many genes, including homeodomain leucine zipper IV transcription factors ROC5 and ROC8, regulating rice leaf rolling have been cloned and functionally analysed. However, the molecular mechanism by which these genes modulate leaf-rolling remains largely elusive. In this study, we demonstrated the transcription activation activity of both ROC8 and ROC5. Overexpressing ROC8 caused adaxially rolled leaves due to decreased number and size of bulliform cells, whereas knockout of ROC8 induced abaxially rolled leaves due to increased number and size of bulliform cells. ROC8 and ROC5 each could form homodimer, but ROC8 interacted preferably with ROC5 to forms a heterodimer. Importantly, we showed that the ROC8-ROC5 heterodimer rather than the homodimer of ROC8 or ROC5 was functional as neither overexpressing ROC8 in the ROC5 mutant nor overexpressing ROC5 in the ROC8-knockout line could rescue the mutant phenotype. This was further partially supported by the identification of a large number of common differentially expressed genes in single and double mutants of roc8 and roc5. ROC8 and ROC5 were functionally additive as the phenotype of abaxially rolled leaves was stronger in the roc5roc8 double mutant than in their single mutants. Our results provide evidence for the role of dimerization of ROC members in regulating leaf rolling of rice.

Integration Analysis of Small RNA and Degradome Sequencing Reveals MicroRNAs Responsive to Dickeya zeae in Resistant Rice.

Rice foot rot disease caused by the pathogen Dickeya zeae (formerly known as Erwinia chrysanthemi pv. zeae), is a newly emerging damaging bacterial disease in China and the southeast of Asia, resulting in the loss of yield and grain quality. However, the genetic resistance mechanisms mediated by miRNAs to D. zeae are unclear in rice. In the present study, 652 miRNAs including osa-miR396f predicted to be involved in multiple defense responses to D. zeae were identified with RNA sequencing. A total of 79 differentially expressed miRNAs were detected under the criterion of normalized reads ≥10, including 51 known and 28 novel miRNAs. Degradome sequencing identified 799 targets predicted to be cleaved by 168 identified miRNAs. Among them, 29 differentially expressed miRNA and target pairs including miRNA396f-OsGRFs were identified by co-expression analysis. Overexpression of the osa-miR396f precursor in a susceptible rice variety showed enhanced resistance to D. zeae, coupled with significant accumulation of transcripts of osa-miR396f and reduction of its target the Growth-Regulating Factors (OsGRFs). Taken together, these findings suggest that miRNA and targets including miR396f-OsGRFs have a role in resisting the infections by bacteria D. zeae.

Examining two sets of introgression lines reveals background-independent and stably expressed QTL that improve grain appearance quality in rice (Oryza sativa L.).

KEY MESSAGE: A novel QTL cluster for appearance quality on Chr07 was identified using reciprocal introgression populations in different locations in China. Two secondary F 2 populations validated QTL with significant effect on appearance quality. Appearance quality (AQ) is the main determinants of market value of rice. Identification of QTL affecting AQ is the prerequisite for efficient improvement of AQ through marker-assisted selection (MAS). Two sets of reciprocal introgression lines derived from indica Minghui 63 and japonica 02428 were used to dissect the stability of QTL affecting five AQ traits, including grain length, grain width, length to width ratio, percentage of grains with chalkiness, and degree of endosperm chalkiness using 4568 bin genotype produced from 58,000 SNPs across five different environments. A total of 41 and 30 main-effect QTL were identified in MH63 and 02428 backgrounds, respectively. Among them, 9 background-independent QTL (BI-QTL) were found. There were also 13 and 10 stable-expressed QTL (SE-QTL) across at least two environments in MH63 and 02428 backgrounds, respectively. Two important BI- and SE-QTL regions (BISERs) including BISER-I harboring qPGWC5, qDEC5, qGW5.1, and qLWR5 on chromosome 5 and BISER-II harboring qGL7, qLWR7, qPGWC7, and qDEC7 on chromosome 7 were identified. The BISER-II was newly reported and validated by two secondary F2 populations in the reciprocal backgrounds. Among 59 epistatic QTL (E-QTL) detected in this study, there were only four SE- but no BI-E-QTL detected in different environments, indicating that genetic background has stronger effect on AQ traits than the environmental factors, especially for percentage of grains with chalkiness (PGWC) and degree of endosperm chalkiness (DEC) with lower heritability. BISER-I and BISER-II harboring many BI- and SE-QTL with favorable alleles from slender grain rice are much important for improvement of rice AQ by MAS.

Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus.

Rice black-streaked dwarf virus (RBSDV) belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA) construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21-24 nt small interfering RNA (siRNA). By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

RGG1, Involved in the Cytokinin Regulatory Pathway, Controls Grain Size in Rice.

Heterotrimeric GTP binding proteins (G proteins) and cytokinin play important roles in regulating plant growth and development. However, little is known about the mechanism by which they coordinate the regulation of grain size in rice. We functionally characterized one gene, RGG1, encoding a type-A Gγ subunit. Strong GUS staining was detected in young panicles and spikelets, suggesting a role for this gene in modulating panicle-related trait development. Overexpression of RGG1 in Nipponbare (NIP) and Wuyunjing 30 (WYJ30) significantly decreased plant height, panicle length and grain length by regulating cell division. However, rgg1 mutants generated by the CRISPR/Cas9 system exhibited no obvious phenotypic differences, which may be due to the extremely low expression level of this gene in vivo. The transcriptomes of young panicles of NIP, the NIP-rgg1-2 mutant and the NIP-OE2 overexpression line were sequenced, and the results showed that many differentially expressed genes (DEGs) were associated with the cytokinin biosynthetic pathway. We confirmed this result by measuring the endogenous cytokinin levels and found that cytokinin content was lower in the overexpression lines. Additionally, increased expression of RGG1 decreased sensitivity to low concentrations of 6-benzylaminopurine (6-BA). Our results reveal a novel G protein-cytokinin module controlling grain size in rice and will be beneficial for understanding the mechanisms by which G proteins regulate grain size and plant development.

Comparative transcriptome analysis reveals molecular response to salinity stress of salt-tolerant and sensitive genotypes of indica rice at seedling stage.

Abiotic stresses, such as salinity, greatly threaten the growth and productivity of plants. Rice (Oryza sativa L.) is one of the most important food crops, as well as a monocot model for genomic research. To obtain a global view of the molecular response to salinity stress, we conducted a leaf transcriptome analysis on rice seedlings. Two cultivars of rice subspecies indica, including the salt-tolerant genotype Xian156 and the salt-sensitive genotype IR28, were used in the present study. Eighteen RNA libraries were obtained from these two genotypes at three timepoints (0 h, 48 h and 72 h) after applying salinity stress. We obtained the reference-guided assembly of the rice transcriptome, which resulted in 1,375 novel genes, including 1,371 annotated genes. A comparative analysis between genotypes and time points showed 5,273 differentially expressed genes (DEGs), of which 286 DEGs were only found in the tolerant genotype. The Disease resistance response protein 206 and TIFY 10 A were differentially expressed, which were validated by quantitative real-time PCR. The differentially expressed genes identified through the mRNA transcriptome, along with the structure, provide a revealing insight into rice molecular response to salinity stress and underlie the salinity tolerance mechanism between genotypes.

Overexpressing CYP71Z2 enhances resistance to bacterial blight by suppressing auxin biosynthesis in rice.

BACKGROUND: The hormone auxin plays an important role not only in the growth and development of rice, but also in its defense responses. We've previously shown that the P450 gene CYP71Z2 enhances disease resistance to pathogens through regulation of phytoalexin biosynthesis in rice, though it remains unclear if auxin is involved in this process or not. METHODOLOGY AND PRINCIPAL FINDINGS: The expression of CYP71Z2 was induced by Xanthomonas oryzae pv. oryzae (Xoo) inoculation was analyzed by qRT-PCR, with GUS histochemical staining showing that CYP71Z2 expression was limited to roots, blades and nodes. Overexpression of CYP71Z2 in rice durably and stably increased resistance to Xoo, though no significant difference in disease resistance was detected between CYP71Z2-RNA interference (RNAi) rice and wild-type. Moreover, IAA concentration was determined using the HPLC/electrospray ionization/tandem mass spectrometry system. The accumulation of IAA was significantly reduced in CYP71Z2-overexpressing rice regardless of whether plants were inoculated or not, whereas it was unaffected in CYP71Z2-RNAi rice. Furthermore, the expression of genes related to IAA, expansin and SA/JA signaling pathways was suppressed in CYP71Z2-overexpressing rice with or without inoculation. CONCLUSIONS AND SIGNIFICANCE: These results suggest that CYP71Z2-mediated resistance to Xoo may be via suppression of IAA signaling in rice. Our studies also provide comprehensive insight into molecular mechanism of resistance to Xoo mediated by IAA in rice. Moreover, an available approach for understanding the P450 gene functions in interaction between rice and pathogens has been provided.