With the passage of time and the advancement of technology, crop breeding has gone through generations from 1.0 to 4.0 and is now moving towards generation 5.0. Although the 3.0 and 4.0 generations of breeding have received extensive attention, only hybrid breeding of the 2.0 generation can enable the parents to achieve genome-wide recombination, resulting in a large number of complex and unpredictable interactions within and between genes, which may be the basis for the emergence of breakthrough traits. Thus hybrid breeding still holds an important position. However, at present, taking rice as an example, the hybrid breeding operations carried out by the majority of breeders may still have issues that need improvement in terms of scientificity and efficiency. In light of the current situation, in order to select high-yielding, high-quality, and multi-resistant varieties, and to overcome the homogenization of varieties, hybrid rice breeding should pay attention to the following aspects. Firstly, the breeding goals should be combined with the local natural conditions and effectively coordinate the combination of advantageous traits. Only in this way can the high-yield, high-quality and highly-resistant high-level goals be achieved, so as to break through the homogenization of varieties. Secondly, because the F1 generation combines the superior traits of both parents and has certain hybrid vigor, it may be the best-performing generation of the same combination. If F1 performs poorly overall, it is difficult for its offspring to produce the expected types that meet the breeding goals. Therefore, this generation should be selected as a key generation, which is conducive to significantly improving the efficiency of breeding. Thirdly, in the early stage of breeding, the main task is to promote generations. To enhance the breeding efficiency, direct seeding should be adopted, which can save land and resources. During the breeding process, the current generation should be combined with the early-generation tests to increase predictability and further eliminate combinations to improve the breeding efficiency. Fourth, during the high-generation selection process, after field selecting, the panicle traits of the combinations should be further compared indoors to select the optimal combination, so as to achieve the best from the best. Finally, the intelligent varieties of the 5.0 generation of breeding are those that can adapt to the ecological and biological factors of the wide range of environments, and can meet the production needs with wide adaptability. Due to the complexity of the environmental conditions for crop growth, it is necessary to conduct extensive and long-term identification of the varieties to achieve the breeding goals. In conclusion, by optimizing the field operations and selection techniques in hybrid breeding, the breeding efficiency will be significantly enhanced, laying the foundation for the selection of breakthrough varieties.
【Objective】 Rapid on-site screening of genetically modified (GM) crops is crucial for effective biosafety regulation. To overcome the limitations of current detection methods, such as equipment dependency and operational complexity, this study developed a closed-tube detection system by integrating recombinase polymerase amplification (RPA) with split DNAzyme (MNAzyme). The system enables rapid, sensitive, and on-site detection of the GM soybean event DBN9004, supporting regulatory compliance and industrial safety management. 【Method】 Using GM soybean DBN9004 and its non-GM counterpart Jack as experimental materials, we firstly identified event-specific sequences for target detection through bioinformatics analysis. Then a recombinant plasmid (9004P) was constructed as a standard template. An asymmetric RPA system was designed to efficiently amplify the target sequence while generating abundant single-stranded DNA (ssDNA) products for MNAzyme activation. Critical reaction parameters were systematically optimized, including reaction temperature (35-60 ℃), probe concentration (125-1 000 nmol·L-1), and RPA primer ratios (10 000﹕10 000 nmol·L-1-10 000﹕31.25 nmol·L-1). Sensitivity assessment was evaluated using gradient-diluted plasmids (8×10-1-8×105 copies/μL), while specificity evaluation was verified against ten GM crop lines (GTS40-3-2, ZH10-6, etc.). Field samples (n=13) were tested and compared with qPCR results. 【Result】 The method demonstrated exceptional sensitivity (8 copies/reaction), good repeatability (RSD=4.44%) and reproducibility (RSD=5.75%), absolute specificity for DBN9004 with no cross-reactivity against ten prevalent GM soybean varieties. Field testing demonstrated perfect concordance (100%) with qPCR results (n=13). 【Conclusion】 This study implemented an asymmetric RPA strategy to efficiently generate target-specific ssDNA amplicons. The resulting ssDNA products demonstrate specific binding affinity for pre-engineered split DNAzyme subunits (A/B), triggering their activation and subsequent continuous cleavage of fluorophore-quencher labeled substrate probes. Leveraging this molecular mechanism, we established a novel RPA-MNAzyme integrated platform for rapid and reliable detection of genetically modified soybean event DBN9004. By combining asymmetric RPA with MNAzyme cascade amplification, the method achieves dual-specificity recognition and signal enhancement. The closed-tube design prevents aerosol contamination, while the dual-mode output system accommodates both laboratory and on-site screening needs.
【Objective】 The upper reaches of the Yangtze River represent one of China’s major rapeseed-producing regions, playing a pivotal role in ensuring the national supply of edible vegetable oil and improving the self-sufficiency rate of oil crops. However, the region is characterized by complex and variable climatic conditions, and traditional genetic improvement evaluation methods are highly susceptible to environmental interference, making it difficult to accurately track trends in varietal genetic potential. This study aimed to establish a method for tracing genetic improvement using annually top-yielding lines as representatives, thereby systematically revealing the genetic progress trajectory and agronomic trait evolution of rapeseed lines in the upper reaches of the Yangtze River from 2004 to 2023. The goal was to clarify the synergistic regulatory mechanisms of key agronomic traits in high-yield lines and provide theoretical support for high-yield and stable-yield rapeseed breeding. 【Method】 Annual top-yielding lines from the National Winter Rapeseed Regional Trials conducted in the upper reaches of the Yangtze River from 2004 to 2023 were selected as the study objects. A mixed linear model was used to separate environmental effects via the best linear unbiased prediction (BLUP) model. Linear regression, Pearson’s correlation analysis, standardized path analysis, and principal component analysis (PCA) were integrated to comprehensively assess yield genetic progress trends and trait variation patterns over the past two decades, and to construct a multi-trait synergistic regulation network. 【Result】 Both actual yield and BLUP-based yield of the rapeseed lines showed a significant upward trend from 2004 to 2023. The improvement of traits in high-yield lines exhibited clear stage-specific changes: from 2004-2013, breeding strategies emphasized compact plant type, with significant reductions in branching number, siliques per plant, and whole growth period; from 2014-2023, strategies shifted toward seed number type, with marked increases in siliques per plant and thousand-seed weight. Correlation and path analyses revealed that yield per plant is the core direct driving factor for enhancing population yield, while silique number per plant and branching number primarily contribute to population yield through indirect pathways via their effects on yield per plant. PCA revealed that the first five principal components all had eigenvalues greater than 1, cumulatively explaining 76% of the total variance. The PC1 axis, predominantly characterized by structural traits such as silique number per plant, branching number, and plant height, accounted for 29% of the variance, representing the primary dimension underlying inter-varietal differentiation, indicating a breeding trend from “single-trait breakthroughs” to “multi-factor synergy”. 【Conclusion】 The breeding focus for rapeseed in the upper Yangtze River has shifted from early-stage optimization of plant architecture toward late-stage enhancement of seed number. The study identified a high-yield model centered on yield per plant, supported by the coordinated improvement of branching number and siliques per plant, with balanced allocation to seed size traits. The lack of promotion area for high-yield varieties in the upper reaches of the Yangtze River from 2004 to 2013 indicates that the breeding direction in this region should comprehensively consider oil production and lodging resistance, in order to achieve sustained break throughs in rapeseed yield under different ecological and management conditions in the upper reaches of the Yangtze River.
