To analyze the genetic diversity relationship of phenotypic traits in forage oat germplasm resources worldwide, conduct Cluster analysis and Comprehensive evaluation, explore excellent materials, and provide Material basis and Technical support for Germplasm creation and Breeding utilization of forage oats.

Using 244 collected domestic and foreign forage oat germplasm as experimental materials, they were planted in Guyuan City, Ningxia from 2023 to 2024, and 11 Phenotypic traits including plant height, stem diameter, tiller number, leaf number, leaf length, leaf width, ear length, whorled layers number, number of spikelets, fresh weight, and hay weight were systematically measured. Multiple evaluation methods such as correlation analysis, principal component analysis, membership function, and hierarchical cluster analysis were comprehensively used for multi-dimensional evaluation.

Phenotypic variant analysis showed that the coefficient of variation of the 11 traits ranged from 12.11% to 42.69%, among which tiller number, leaf length, and number of spikelets had higher coefficient of variation, which were 42.69%, 32.90%, and 32.77% respectively; The genetic diversity index ranged from 1.408 to 2.077, among which number of spikelets, stem diameter, and hay weight had higher genetic diversity index, which were 2.077, 2.075, and 2.074 respectively; Correlation analysis revealed that there were 41 pairs of significant or extremely significant relationships among the 11 traits, and the indicators with higher correlation with hay weight of forage oats were fresh weight, leaf number, leaf width, whorled layers number, plant height, and number of spikelets in sequence; The principal component analysis showed that the contribution rate of six principal components were extracted, which were 28.980%, 14.833%, 12.494%, 9.556%, 7.495%, and 6.850% in sequence, with the Cumulative contribution rate reaching 80.209%; Hierarchical cluster analysis divided the 244 germplasm resources into 5 Groups. Among them, the germplasm resources in Group had thick stem diameter, long ear length, and many whorled layers Ⅱ number, belonging to lodging-resistant or high yield materials; The germplasm in Group Ⅲ and Ⅳ had high plant height, long leaf length, and many tiller number, belonging to high yield forage materials; The germplasm in Group Ⅴ had multiple leaves, wide Leaf width, many number of spikelets, and high fresh and hay weight, belonging to high-quality forage type and high grain yield materials. The Comprehensive phenotypic evaluation value (F value) of germplasm resources was obtained through analytical methods such as membership function, and 20 accessions of Core germplasm resources with excellent comprehensive traits were screened out.

The results revealed that the 244 forage oat germplasm resources possess rich phenotypic genetic variation and diversity; Cluster analysis divided the 244 germplasm resources into 5 different groups, which can be selected and utilized according to the excellent characteristics of different germplasm resources and breeding objectives; 20 accessions of forage oat germplasm resources with excellent comprehensive traits, such as As166, As129, As163, As131 and As80 were screened out using F value.

To identify and evaluate the drought resistance of alfalfa germplasm resources, screen out different drought-resistant alfalfa germplasm materials, and lay a foundation for further development of drought-resistant alfalfa resources creation and breeding utilization.

In this study, A total of 111 alfalfa germplasm resources were used as experimental materials, and drought stress conditions were simulated using a 13% PEG-6000 solution. Two treatments were established: drought stress (13% PEG-6000) and a control (distilled water). Drought tolerance at germination stage were comprehensively evaluated by the comprehensive drought resistance coefficient (CDC value) and drought resistance coefficient (D value) using nine indicators, including germination potential, germination rate, germination index, vitality index, promptness index, root length, bud length, fresh weight and dry weight. Meanwhile, single drought tolerance coefficients, correlation analysis, principal component analysis and stepwise regression analysis were conducted to identify the key indicators influencing drought resistance during germination.

Analysis of the single drought tolerance coefficients for the nine indicators showed that vitality index, fresh weight, and promptness index were key indicators for screening drought resistance at the germination stage. The single drought tolerance coefficients of the indicators had the highest correlation among germination index and promptness index, germination rate, vitality index, with coefficients of 0.9838, 0.9495 and 0.9338, respectively. Principal component analysis transformed the nine indicators into three principal components with a cumulative contribution of 87.287%. Drought resistance of alfalfa at germination stage was identified using two methods with CDC value and D value, and it was found that the evaluation results of the two methods were highly consistent; however, the D value method was more comprehensive, reliable, and accurate, as it used the weighting coefficients to reflect the degree of influence of each indicator to the overall drought resistance of the varieties. Based on the D value, the 111 alfalfa germplasm resources were clustered into five categories: ClassⅠ(strongest drought resistance, 1 accession), Class Ⅱ (strong drought resistance, 5 accessions), Class Ⅲ (moderate drought resistance, 55 accessions), Class Ⅳ (weak drought resistance, 37 accessions), and Class Ⅴ (drought-sensitive, 13 accessions).

The D value evaluation method was found to be the most reliable for assessing drought resistance in alfalfa during the germination stage; The study identified AG37 as the strongest drought resistance germplasm, along with five strong drought resistance accessions, including AG19, AG5, AG13 and other. Vitality index and promptness index were determined to be the most suitable indicators for evaluating drought resistance in alfalfa at the germination stage.

Branching is a significant factor influencing alfalfa yield, and the SPL family of transcription factors represents a crucial class of regulatory genes involved in branching (tillering) development in a diverse range of plants. The objective of this reserch is to investigate the biological function of MsSPL17 in alfalfa and to elucidate the role of MsSPL17 in regulating the development of alfalfa meristems. This will provide a valuable reference for alfalfa high-yield biological breeding.

Bioinformatics methods were used to anlyze MsSPL17 sequences and also constructing a phylogenetic tree. The tissue expression specificity of MsSPL17 in alfalfa was analyzed by real-time fluorescence quantitative PCR (qPCR). The subcellular localization of MsSPL17 protein was determined by tobacco transient expression system, and the transcriptional self-activation activity of MsSPL17 was verified. The transgenic alfalfa was obtained by Agrobacterium-mediated transformation and phenotypic analysis was carried out. Transcriptome analysis was utilized to screen for differentially expressed genes in transgenic lines and validate them for further research.

MsSPL17 contained an open reading frame of 1 011 bp, encoding a protein composed of 366 amino acids, belonging to the SBP protein family. Phylogenetic analysis showed that the evolution of MsSPL17 and its homologous genes was highly similar to the differentiation of species, indicating that it is a functional conserved gene. MsSPL17 expressed in all tissues, including stems, nodes, leaves and tops during the critical period of alfalfa growth and development, implied the necessary regulating function of this gene in alfalfa branching. Subcellular localization assay showed that MsSPL17 protein was localized in the nucleus. Transcriptional self-activation assay showed that MsSPL17 did not have self-activation activity and could be used in interacting proteins screening. MsSPL17 transgenic silenced lines exhibited a notable phenotype, including an increase in branch number and stem node number, a reduction in internode length, and an enhancement in nutritional quality.

MsSPL17 was successfully cloned, it expressed in key tissues of alfalfa branching development. The protein encoded by MsSPL17 was localized in the nucleus and demonstrated no transcriptional self-activation activity. Transgenic lines exhibiting multi-branching traits were obtained, and the number of branches increased significantly in yield, while the crude protein content increased in quality.