A new three-dimensional (3D) molecularly imprinted polymer fluoroprobe based on green-red dual-emission signals of carbon quantum dots and self-polymerization of dopamine (CDs@PDA-MIPs) for sensitive detection of nifedipine.

Nifedipine (NIF), as one of the dihydropyridine calcium channel blockers, is widely used in the treatment of hypertension. However, misuse or ingestion of NIF can result in serious health issues such as myocardial infarction, arrhythmia, stroke, and even death. It is essential to design a reliable and sensitive detection method to monitor NIF. In this work, an innovative molecularly imprinted polymer dual-emission fluorescent sensor (CDs@PDA-MIPs) strategy was successfully designed for sensitive detection of NIF. The fluorescent intensity of the probe decreased with increasing NIF concentration, showing a satisfactory linear relationship within the range 1.0 × 10-6 M ~ 5.0 × 10-3 M. The LOD of NIF was 9.38 × 10-7 M (S/N = 3) in fluorescence detection. The application of the CDs@PDA-MIPs in actual samples such as urine and Qiangli Dingxuan tablets has been verified, with recovery ranging from 97.8 to 102.8% for NIF. Therefore, the fluorescent probe demonstrates great potential as a sensing system for detecting NIF.

Comprehensive evaluation of resistance effects of pyramiding lines with different broad-spectrum resistance genes against Magnaporthe oryzae in rice (Oryza sativa L.).

BACKGROUND: Broad-spectrum resistance gene pyramiding helps the development of varieties with broad-spectrum and durable resistance to M. oryzae. However, detailed information about how these different sources of broad-spectrum resistance genes act together or what are the best combinations to achieve broad-spectrum and durable resistance is limited. RESULTS: Here a set of fifteen different polygene pyramiding lines (PPLs) were constructed using marker-assisted selection (MAS). Using artificial inoculation assays at seedling and heading stage, combined with natural induction identification under multiple field environments, we evaluated systematically the resistance effects of different alleles of Piz locus (Pigm, Pi40, Pi9, Pi2 and Piz) combined with Pi1, Pi33 and Pi54, respectively, and the interaction effects between different R genes. The results showed that the seedling blast and panicle blast resistance levels of PPLs were significantly higher than that of monogenic lines. The main reason was that most of the gene combinations produced transgressive heterosis, and the transgressive heterosis for panicle blast resistance produced by most of PPLs was higher than that of seedling blast resistance. Different gene pyramiding with broad-spectrum R gene produced different interaction effects, among them, the overlapping effect (OE) between R genes could significantly improve the seedling blast resistance level of PPLs, while the panicle blast resistance of PPLs were remarkably correlated with OE and complementary effect (CE). In addition, we found that gene combinations, Pigm/Pi1, Pigm/Pi54 and Pigm/Pi33 displayed broad-spectrum resistance in artificial inoculation at seedling and heading stage, and displayed stable broad-spectrum resistance under different disease nursery. Besides, agronomic traits evaluation also showed PPLs with these three gene combinations were at par to the recurrent parent. Therefore, it would provide elite gene combination model and germplasms for rice blast resistance breeding program. CONCLUSIONS: The development of PPLs and interaction effect analysis in this study provides valuable theoretical foundation and innovative resources for breeding broad-spectrum and durable resistant varieties.

Identification of Genes Related to Cold Tolerance and a Functional Allele That Confers Cold Tolerance.

Cold stress is a major factor limiting rice (Oryza sativa) production worldwide, especially at the seedling and booting stages. The identification of genes associated with cold tolerance (CT) in rice is important for sustainable food production. Here, we report the results of a genome-wide association study to identify the genetic loci associated with CT by using a 1,033-accession diversity panel. We identified five CT-related genetic loci at the booting stage. Accessions carrying multiple cold-tolerant alleles displayed a higher seed-setting rate than did accessions that had no cold-tolerant alleles or carried a single allele. At the seedling stage, eight genetic loci related to CT have been identified. Among these, LOC_Os10g34840 was identified as the candidate gene for the qPSR10 genetic locus that is associated with CT in rice seedlings. A single-nucleotide polymorphism (SNP), SNP2G, at position 343 in LOC_Os10g34840 is responsible for conferring CT at the seedling stage in rice. Further analysis of the haplotype network revealed that SNP2G was present in 80.08% of the temperate japonica accessions but only 3.8% of the indica ones. We used marker-assisted selection to construct a series of BC4F3 near-isogenic lines possessing the cold-tolerant allele SNP2G When subjected to cold stress, plants carrying SNP2G survived better as seedlings and showed higher grain weight than plants carrying the SNP2A allele. The CT-related loci identified here and the functional verification of LOC_Os10g34840 will provide genetic resources for breeding cold-tolerant varieties and for studying the molecular basis of CT in rice.

Development and Evaluation of Near-Isogenic Lines with Different Blast Resistance Alleles at the Piz Locus in japonica Rice from the Lower Region of the Yangtze River, China.

Rice blast, caused by Magnaporthe oryzae, threatens rice production in most of the rice-growing areas in China, especially in regions that have grown Oryza sativa subsp. japonica in recent years. The use of resistance genes is the most effective and economical approach for blast control. In our study, a set of six near-isogenic lines (NIL) were developed by introgression of six resistance alleles of the Piz locus (Pi2, Pigm, Pi40, Pi9, Piz, and Pizt) into a blast-susceptible, high-yielding, high-quality japonica '07GY31' via marker-assisted backcross breeding. Artificial inoculation using 144 M. oryzae isolates collected from the lower region of the Yangtze River, China, revealed that most of the NIL, including NIL-Pi2, NIL-Pigm, NIL-Pi40, NIL-Pi9, and NIL-Pizt, exhibited broad-spectrum resistance against rice blast at the seedling stage, with resistance frequencies (RF) of 93.06 to 98.61%. NIL-Piz was an exception, with an RF of 21.53%, which was slightly higher than the recurrent parent 07GY31. NIL-Pi40 and NIL-Pigm had broad-spectrum resistance (RF of 93.33 and 71.67%, respectively) at the heading stage following inoculation of 60 isolates of M. oryzae. Field trials with artificial inoculation at the seedling and heading stage showed that NIL-Pigm and NIL-Pi40 were highly resistant in four locations under high disease pressure. NIL-Pizt showed effective resistance in three locations from Zhejiang and Jiangsu Provinces. This study shows that O. sativa subsp. japonica alleles of the Piz locus confer resistance to M. oryzae, and provides an effective method to enhance seedling and panicle blast resistance in rice plants in the lower region of the Yangtze River, China.

Improving of Rice Blast Resistances in Japonica by Pyramiding Major R Genes.

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is a major constraint to rice production worldwide. In this study, we developed monogenic near-isogenic lines (NILs) NIL Pi9, NIL Pizt , and NIL Pi54 carrying genes Pi9, Pizt, and Pi54, respectively, by marker assisted backcross breeding using 07GY31 as the japonica genetic background with good agronomic traits. Polygene pyramid lines (PPLs) PPL Pi9+Pi54 combining Pi9 with Pi54, and PPL Pizt+Pi54 combining Pizt with Pi54 were then developed using corresponding NILs with genetic background recovery rates of more than 97%. Compared to 07GY31, the above NILs and PPLs exhibited significantly enhanced resistance frequencies (RFs) for both leaf and panicle blasts. RFs of both PPLs for leaf blast were somewhat higher than those of their own parental NILs, respectively, and PPL Pizt+Pi54 exhibited higher RF for panicle blast than NIL Pizt and NIL Pi54 (P < 0.001), hinting an additive effect on the resistance. However, PPL Pi9+Pi54 exhibited lower RF for panicle blast than NIL Pi9 (P < 0.001), failing to realize an additive effect. PPL Pizt+Pi54 showed higher resistant level for panicle blast and better additive effects on the resistance than PPL Pi9+Pi54. It was suggested that major R genes interacted with each other in a way more complex than additive effect in determining panicle blast resistance levels. Genotyping by sequencing analysis and extreme-phenotype genome-wide association study further confirmed the above results. Moreover, data showed that pyramiding multiple resistance genes did not affect the performance of basic agronomic traits. So the way to enhance levels of leaf and panicle blast resistances for rice breeding in this study is effective and may serve as a reference for breeders. Key Message: Resistant levels of rice blast is resulted from different combinations of major R genes, PPL Pizt+Pi54 showed higher resistant level and better additive effects on the panicle blast resistance than PPL Pi9+Pi54.

Combination Patterns of Major R Genes Determine the Level of Resistance to the M. oryzae in Rice (Oryza sativa L.).

Rice blast caused by Magnaporthe oryzae is the most devastating disease of rice and poses a serious threat to world food security. In this study, the distribution and effectiveness of 18 R genes in 277 accessions were investigated based on pathogenicity assays and molecular markers. The results showed that most of the accessions exhibited some degree of resistance (resistance frequency, RF >50%). Accordingly, most of the accessions were observed to harbor two or more R genes, and the number of R genes harbored in accessions was significantly positively correlated with RF. Some R genes were demonstrated to be specifically distributed in the genomes of rice sub-species, such as Pigm, Pi9, Pi5 and Pi1, which were only detected in indica-type accessions, and Pik and Piz, which were just harbored in japonica-type accessions. By analyzing the relationship between R genes and RF using a multiple stepwise regression model, the R genes Pid3, Pi5, Pi9, Pi54, Pigm and Pit were found to show the main effects against M. oryzae in indica-type accessions, while Pita, Pb1, Pik, Pizt and Pia were indicated to exhibit the main effects against M. oryzae in japonica-type accessions. Principal component analysis (PCA) and cluster analysis revealed that combination patterns of major R genes were the main factors determining the resistance of rice varieties to M. oryzae, such as 'Pi9+Pi54', 'Pid3+Pigm', 'Pi5+Pid3+Pigm', 'Pi5+Pi54+Pid3+Pigm', 'Pi5+Pid3' and 'Pi5+Pit+Pid3' in indica-type accessions and 'Pik+Pib', 'Pik+Pita', 'Pik+Pb1', 'Pizt+Pia' and 'Pizt+Pita' in japonica-type accessions, which were able to confer effective resistance against M. oryzae. The above results provide good theoretical support for the rational utilization of combinations of major R genes in developing rice cultivars with broad-spectrum resistance.

Fine mapping of the qLOP2 and qPSR2-1 loci associated with chilling stress tolerance of wild rice seedlings.

KEY MESSAGE: Using leaf osmotic potential and plant survival rate as chilling-tolerant trait indices, we identified two major quantitative trait loci qLOP2 and qPSR2 - 1 (39.3-kb region) and Os02g0677300 as the cold-inducible gene for these loci. Chilling stress tolerance (CST) at the seedling stage is an important trait affecting rice production in temperate climate and high-altitude areas. To identify quantitative trait loci (QTLs) associated with CST, a mapping population consisting of 151 BC(2)F(1) plants was constructed by using chilling-tolerant Dongxiang wild rice (Oryza rufipogon Griff.) as a donor parent and chilling-sensitive indica as a recurrent parent. With leaf osmotic potential (LOP) and plant survival rate (PSR) as chilling-tolerant trait indexes, two major QTLs, qLOP2 (LOD = 3.8) and qPSR2-1 (LOD = 3.3), were detected on the long arm of chromosome 2 by composite interval mapping method in QTL Cartographer software, which explained 10.1 and 12.3% of the phenotypic variation, respectively. In R/QTL analyzed result, their major effects were also confirmed. Using molecular marker RM318 and RM106, qLOP2 and qPSR2-1 have been introgressed into chilling-sensitive varieties (93-11 and Yuefeng) by marker-assisted selection procedure (MAS), which resulted in 16 BC(5)F(3) BILs that chilling tolerance have significantly enhanced compare with wild-type parents (P < 0.01). Therefore, two large segregating populations of 11,326 BC(4)F(2) and 8,642 BC(4)F(3) were developed to fine mapping of qLOP2 and qPSR2-1. Lastly, they were dissected to a 39.3-kb candidate region between marker RM221 and RS8. Expression and sequence analysis results indicated that Os02g0677300 was a cold-inducible gene for these loci. Our study provides novel alleles for improving rice CST by MAS and contributes to the understanding of its molecular mechanisms.

Fine mapping of qRC10-2, a quantitative trait locus for cold tolerance of rice roots at seedling and mature stages.

UNLABELLED: Cold stress causes various injuries to rice seedlings in low-temperature and high-altitude areas and is therefore an important factor affecting rice production in such areas. In this study, root conductivity (RC) was used as an indicator to map quantitative trait loci (QTLs) of cold tolerance in Oryza rufipogon Griff., Dongxiang wild rice (DX), at its two-leaf stage. The correlation coefficients between RC and the plant survival rate (PSR) at the seedling and maturity stages were -0.85 and -0.9 (P = 0.01), respectively, indicating that RC is a reliable index for evaluating cold tolerance of rice. A preliminary mapping group was constructed from 151 BC2F1 plants using DX as a cold-tolerant donor and the indica variety Nanjing 11 (NJ) as a recurrent parent. A total of 113 codominant simple-sequence repeat (SSR) markers were developed, with a parental polymorphism of 17.3%. Two cold-tolerant QTLs, named qRC10-1 and qRC10-2 were detected on chromosome 10 by composite interval mapping. qRC10-1 (LOD = 3.1, RM171-RM1108) was mapped at 148.3 cM, and qRC10-2 (LOD = 6.1, RM25570-RM304) was mapped at 163.3 cM, which accounted for 9.4% and 32.1% of phenotypic variances, respectively. To fine map the major locus qRC10-2, NJ was crossed with a BC4F2 plant (L188-3), which only carried the QTL qRC10-2, to construct a large BC5F2 fine-mapping population with 13,324 progenies. Forty-five molecular markers were designed to evenly cover qRC10-2, and 10 markers showed polymorphisms between DX and NJ. As a result, qRC10-2 was delimited to a 48.5-kb region between markers qc45 and qc48. In this region, Os10g0489500 and Os10g0490100 exhibited different expression patterns between DX and NJ. Our results provide a basis for identifying the gene(s) underlying qRC10-2, and the markers developed here may be used to improve low-temperature tolerance of rice seedling and maturity stages via marker-assisted selection (MAS). KEY MESSAGE: With root electrical conductivity used as a cold-tolerance index, the quantitative trait locus qRC10-2 was fine mapped to a 48.5-kb candidate region, and Os10g0489500 and Os10g0490100 were identified as differently expressed genes for qRC10-2.

Screening for and genetic analysis on T-DNA-inserted mutant pool in rice.

T-DNA tagging technique has provided a powerful strategy for identifying new functional genes in plants, and the key for success is the discovery of T-DNA-inserted mutants with changed phenotype. In this study, we screened 4,416 rice T1 tagged lines generated by enhancer trap system integrated with GLL4/VP16-UAS elements from two transformed parents, ZH11 and ZH15. We found many lines showed obvious morphological mutations, including two types--fake-homozygous mutation and separating mutation. The mutation phenotype was related to 14 kinds of trait such as plant height, heading date, leaf shape, leaf color, tiller number, panicle shape, spikelet number, grain shape, disease-like mutation, male sterility, awn, and so on. Among them, plant height, heading date, leaf color and male sterility had a comparatively high mutation frequency (over 1%). The mutation frequency of plant height and leaf color had no significant change between different years or transformed parents, but the frequency of heading date and male sterility varied greatly, suggesting that environment had a great effect on the expression of latter two traits. By conducting continuously co-segregating analyses in T1 and T2 generation, we identified 3 T-DNA-inserted mutants with malformed panicle or spikelets, which would provide a base for cloning correlative functional genes. At the same time, we selected randomly 42 lines with mutation phenotype and obtained 40 flanking sequences from 39 tagged lines by plasmid rescue or TAIL-PCR, of which, 26 were vector backbone sequence, 14 had good identity to rice genome sequence. The BlastN result showed the T-DNA preferentially integrated into protein-coding region in plants.